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Archive for the ‘Cardiac Stem Cells’ Category

Death in Cellectis off-the-shelf CAR-T trial triggers FDA hold – FierceBiotech

The FDA has put a phase 1 trial of Cellectis off-the-shelf CAR-T therapy UCARTCS1A on clinical hold after learning of a death in the study. Cellectis said the multiple myeloma patient suffered a cardiac arrest after receiving the highest dose of the anti-CS1 allogeneic CAR-T.

Before joining the Cellectis trial, the patient underwent multiple prior lines of treatment, including with autologous CAR-T cells, without success. In the Cellectis trial, the patient was the first person to receive the higher, 3 million cells per kilogram dose of UCARTCS1A. The patient experienced cytokine release syndrome of undisclosed severity and died of a cardiac arrest 25 days after treatment.

The FDA has placed the trial on clinical hold while Cellectis evaluates the case. According to Cellectis, plans were already afoot to expand the lower, 1 million cells per kilogram dose cohort before the patient death. Preliminary data suggest 1 million cells per kilogram may be the phase 2 dose.

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There are signs the lower dose also has some safety issues. Analysts at Jefferies think investigators gave one or more of the three low-dose patients rituximab to activate the CAR-T safety switch. Work is underway to update the phase 1 protocol to mitigate the potential risks posed by UCARTCS1A.

The modifications may include increased monitoring of parameters related to cytokines. The Jefferies analysts think Cellectis should exclude patients previously treated with anti-BCMA CAR-Ts, such as Johnson & Johnsons JNJ-4528, due to risks related to back-to-back rounds of lymphodepletion, but note that management at the biotech think it is important to enroll that pre-treated population.

In a follow-up note, the analysts identified the use of cyclophosphamide, a chemotherapy drug, in the lymphodepletion regimen as a potential cause of the cardiac arrest. The argument is based on a 2017 paper that describes the case of a patient who died of acute heart failure after receiving a high dose of cyclophosphamide as part of an autologous stem cell transplantation treatment.

Many patients receive cyclophosphamide without suffering cardiac complications, but the analysts see reasons to think the subject enrolled in the Cellectis trial may have been at higher risk. Notably, prior exposure may increase risk, according to the analysts, suggesting the patients previous round of lymphodepletion may have been a factor.

Even if cyclophosphamide is at the heart of the problem, the analysts still think the UCARTCS1A dose is a contributing factor. With patients in the low-dose cohort also experiencing adverse events, the analysts see dosing at below 1 million cells per kilogram as one possible outcome of the situation.

Shares in Cellectis fell 13% in after-hours trading following news of the clinical hold. The value of Allogene Therapeutics, which licensed CAR-T assets that originated at Cellectis, held steady, likely reflecting a belief that the safety issue is limited to UCARTCS1A.

The Jefferies analysts see little or no read-through to other allogeneic programs, noting that the UCARTCS1A trial started at a higher dose than Cellectis two other clinical programs and that Allogene is testing several lymphodepletion regimens. The FDA placed a clinical trial of another Cellectis CAR-T, UCART123, on hold in 2017 after a patient died, but cleared it to resume months later.

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Death in Cellectis off-the-shelf CAR-T trial triggers FDA hold - FierceBiotech

Could induced pluripotent stem cells be the breakthrough genetics has been waiting for? – The New Economy

Embryonic stem cells. The ethical issues associated with stem cell research could be resolved through the use of induced pluripotent stem cells, which are derived from fully committed and differentiated cells of the adult body

The almost miraculous benefits that stem cells may one day deliver have long been speculated on. Capable of becoming different types of cells, they offer huge promise in terms of transplant and regenerative medicine. It is, however, also a medical field that urges caution one that must constantly battle exaggeration. If stem cells do in fact hold the potential to reverse the ageing process, for example, then such breakthroughs remain many years away.

Recently, though, the field has had cause for excitement. In 2006, Japanese researcher Shinya Yamanaka discovered that mature cells could be reprogrammed to become pluripotent, meaning they can give rise to any cell type of the body. In 2012, the discovery of these induced pluripotent stem cells (iPSCs) saw Yamanaka and British biologist John Gurdon awarded the Nobel Prize in Physiology or Medicine. Since then, there has been much talk regarding the potential iPSCs possess, not only for the world of medicine, but for society more generally, too.

A big stepHistorically, one of the major hurdles preventing further research into stem cells has been an ethical one. Until the discovery of iPSCs, embryonic stem cells (ESCs) represented the predominant area of research, with cells being taken from preimplantation human embryos. This process, however, involves the destruction of the embryo and, therefore, prevents the development of human life. Due to differences in opinion over when life is said to begin during embryonic development, stem cell researchers face an ethical quandary.

The promise of significant health benefits and new revenue streams has led some clinics to offer unproven stem cell treatments to individuals

With iPSCs, though, no such dilemmas exist. IPSCs are almost identical to ESCs but are derived from fully committed and differentiated cells of the adult body, such as a skin cell. Like ESCs, iPSCs are pluripotent and, as they are stem cells, can self-renew and differentiate, remaining indefinitely propagated and retaining the ability to give rise to any human cell type over time.

One important distinction to make is that both ESCs and iPSCs do not exist in nature, Vittorio Sebastiano, Assistant Professor (Research) of Obstetrics and Gynaecology (Reproductive and Stem Cell Biology) at Stanford Universitys Institute for Stem Cell Biology and Regenerative Medicine, told The New Economy. They are both beautiful laboratory artefacts. This means that at any stage of development, you cannot find ESCs or iPSCs in the developing embryo, foetus or even in the postnatal or adult body. Both ESCs and iPSCs can only be established and propagated in the test tube.

The reason neither ESCs nor iPSCs can be found in the body is that they harbour the potential to be very dangerous. As Sebastiano explained, these cells could spontaneously differentiate into tumorigenic masses because of their intrinsic ability to give rise to any cell type of the body. Over many years of research, scientists have learned how to isolate parts of the embryo (in the case of ESCs) and apply certain culture conditions that can lock cells in their proliferative and stem conditions. The same is true for iPSCs.

To create iPSCs, scientists take adult cells and exogenously provide a cocktail of embryonic factors, known as Yamanaka factors, for a period of two to three weeks. If the expression of such factors is sustained for long enough, they can reset the programme of the adult cells and establish an embryonic-like programme.

Turning back the clockThere is already a significant body of research dedicated to how stem cells can be used to treat disease. For example, mesenchymal stem cells (usually taken from adult bone marrow) have been deployed to treat bone fractures or as treatments for autoimmune diseases. It is hoped that iPSCs could hold the key for many more treatments.

Global stem cell market:25.5%Expected compound annual growth rate (2018-24)$467bnExpected market value (2024)

IPSCs are currently utilised to model diseases in vitro for drug screening and to develop therapies that one day will be implemented in people, Sebastiano explained. Given their ability to differentiate into any cell type, iPSCs can be used to differentiate into, for example, neurons or cardiac cells, and study specific diseases. In addition, once differentiated they can be used to test drugs on the relevant cell type. Some groups and companies are developing platforms for cell therapy, and I am personally involved in two projects that will soon reach the clinical stage.

Perhaps the most exciting prospects draw on iPSCs regenerative properties. Over time, cells age for a variety of reasons namely, increased oxidative stress, inflammation and exposure to pollutants or sunlight, among others. All these inputs lead to an accumulation of epigenetic mistakes those that relate to gene expression rather than an alteration of the genetic code itself in the cells, which, over time, results in the aberrant expression of genes, dysfunctionality at different levels, reduced mitochondrial activity, senescence and more besides. Although the epigenetic changes that occur with time may not be the primary cause of ageing, the epigenetic landscape ultimately affects and controls cell functionality.

What we have shown is that, if instead of being expressed for two weeks we express the reprogramming factors for a very short time, then we see that the cells rejuvenate without changing their identity, Sebastiano said. In other words, if you take a skin cell and express the reprogramming genes for two to four days, what you get is a younger skin cell.

By reprogramming a cell into an iPSC, you end up with an embryonic-like cell the reprogramming erases any epigenetic errors. If expressed long enough, it erases the epigenetic information of cell identity, leaving embryonic-like cells that are also young.

Slow and steadyAs with any scientific advancement, financial matters are key. According to Market Research Engine, the global stem cell market is expected to grow at a compound annual growth rate of 25.5 percent between 2018 and 2024, eventually reaching a market value of $467bn. The emergence of iPSCs has played a significant role in shaping these predictions, with major bioscience players, such as Australias Mesoblast and the US Celgene, working on treatments involving this particular type of stem cell.

The business potential around stem cell research is huge, Sebastiano told The New Economy. [Particularly] when it comes to developing cell banks for which we have detailed genetic information and, for example, studying how different drugs are toxic or not on certain genetic backgrounds, or when specific susceptibility mutations are present.

Unfortunately, even as the business cases for iPSC treatments increase, a certain degree of caution must be maintained. The promise of significant health benefits and new revenue streams has led some clinics to offer unproven stem cell treatments to individuals. There have been numerous reports of complications emerging, including the formation of a tumour following experimental stem cell treatment in one particular patient, as recorded in the Canadian Medical Association Journal last year. Such failures risk setting the field back years.

The challenge for researchers now will be one of balance. The potential of iPSCs is huge both in terms of medical progress and business development but can easily be undermined by misuse. Medical advancements, particularly ones as profound as those associated with iPSCs, simply cannot be rushed.

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Could induced pluripotent stem cells be the breakthrough genetics has been waiting for? - The New Economy

New technology May Raise the quality of stem cells Found in regenerative medicine – Microbioz India

Stem cells have been holding great promise for regenerative medicine for ages. In the last decade, many studies have revealed this form of cell, which in Spanish is calledmother cell due to its ability to contribute to various different cell types, may be applied in regenerative medicine to diseases such as muscle and nervous system disorders, among others.

Scientists and stem cell leaders Sir John B. Gurdon and Shinya Yamanaka received the Nobel Prize in Physiology and Medicine in 2012 for this idea.

However, one of the key constraints in the application of these herbal remedies is the caliber of the stem cells that may be made in the lab, which impedes their use for curative purposes.

Currently, a team in the Cell Division and Cancer Group of the Spanish National Cancer Research Centre (CNIO), headed by researcher Marcos Malumbres, has recently developed a fresh, easy and fast technology that enhances in vitro and in vivo the possibility of stem cells to differentiate into adult cells. The study results will be released this week in The EMBO Journal.

In recent years, several protocols have been proposed to obtain reprogrammed stem cells in the laboratory from adult cells, but very few to improve the cells we already have.The method we developed is able to significantly increase the quality of stem cells obtained by any other protocol, thus favouring the efficiency of the production of specialised cell types.Mara Salazar-Roa, Study First Author and Researcher, Centro Nacional de Investigaciones Oncolgicas

Roa is likewise the co-corresponding author of this analysis.

Within this study, the researchers identified an RNA sequence, called microRNA 203, that can be found at the earliest embryonic stages before the embryo implants in the uterus and when stem cells have their highest ability to generate all the different cells.When they added this molecule to stem cells from the laboratory, they discovered that the cells ability to convert into other cell types improved appreciably.

To corroborate them, they used stem cells of both human and murine origin, and of genetically altered mice. The results were so spectacular, both in mouse cells and in human cells

Application of the microRNA for just 5 days boosts the potential of stem cells in most situations we tested and improves their ability to become other specialised cells, even months after being connected with the microRNA. Says Salazar-Roa.

According to the research, cells modified by this new protocol are more efficient in generating functional cardiac cells, opening the doorway to a better generation of different cell types essential for the cure of degenerative disorders.

Malumbres, mind of the CNIO Cell and Cancer Division Group, states:To deliver this asset to the clinic, cooperation with labs or companies that are looking to exploit that technology is now essential in each particular case.

In this circumstance, Salazar-Roa recently participated, in close collaboration with all the CNIOs Innovation group, in prestigious creation programs like IDEA2 International of the Massachusetts Institute of Technology (MIT) and also CaixaImpulse of thisLa Caixa Foundation, where they also obtained funding to start the maturation of the technology.

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New technology May Raise the quality of stem cells Found in regenerative medicine - Microbioz India

Expanded Access Protocol Initiated for Compassionate Use of Remestemcel-L in Children With Multisystem Inflammatory Syndrome Associated With COVID-19…

NEW YORK, July 06, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB) today announced that an expanded access protocol (EAP) has been initiated in the United States for compassionate use of its allogeneic mesenchymal stem cell (MSC) product candidate remestemcel-L in the treatment of COVID-19 infected children with cardiovascular and other complications of multisystem inflammatory syndrome (MIS-C). Patients aged between two months and 17 years may receive one or two doses of remestemcel-L within five days of referral under the EAP.

The protocol was filed with the United States Food and Drug Administration (FDA) and provides physicians with access to remestemcel-L for an intermediate-size patient population1 under Mesoblast’s existing Investigational New Drug (IND) application. According to the FDA, expanded access is a potential pathway for a patient with an immediately life-threatening condition or serious disease or condition to gain access to an investigational medical product for treatment outside of clinical trials when no comparable or satisfactory alternative therapy options are available.

MIS-C is a life-threatening complication of COVID-19 in otherwise healthy children and adolescents that includes massive simultaneous inflammation of multiple critical organs and their vasculature. In approximately 50% of cases this inflammation is associated with significant cardiovascular complications that directly involve heart muscle and may result in decreased cardiac function. In addition, the virus can result in dilation of coronary arteries with unknown future consequences. Recent articles from Europe and the United States have described this disease in detail.2-5

Mesoblast Chief Medical Officer Dr Fred Grossman said: The extensive body of safety and efficacy data generated to date using remestemcel-L in children with graft versus host disease suggest that our cellular therapy could provide a clinically important therapeutic benefit in MIS-C patients, especially if the heart is involved as a target organ for inflammation. Use of remestemcel-L in children with COVID-19 builds on and extends the potential application of this cell therapy in COVID-19 cytokine storm beyond the most severe adults with acute respiratory distress syndrome.”

Remestemcel-L Remestemcel-L is an investigational therapy comprising culture-expanded mesenchymal stem cells derived from the bone marrow of an unrelated donor and is administered in a series of intravenous infusions. Remestemcel-L is believed to have immunomodulatory properties to counteract the inflammatory processes that are implicated in several diseases by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

1.www.clinicaltrials.gov; NCT04456439 2.Lancet2020; May 7. DOI: https://doi.org/10.1016/S0140-6736(20)31094-1 3.Lancet. 2020; (May 13) https://doi.org/10.1016/S0140-6736(20)31103-X 4.https://www.nejm.org/doi/full/10.1056/NEJMoa2021756 5.https://www.nejm.org/doi/full/10.1056/NEJMoa2021680

About Mesoblast Mesoblast Limited (Nasdaq:MESO; ASX:MSB) is a world leader in developing allogeneic (off-the-shelf) cellular medicines. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of commercial products and late-stage product candidates. Mesoblast has a strong and extensive global intellectual property (IP) portfolio with protection extending through to at least 2040 in all major markets. The Company’s proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Mesoblast’s Biologics License Application to seek approval of its product candidate RYONCIL (remestemcel-L) for pediatric steroid-refractory acute graft versus host disease (acute GVHD) has been accepted for priority review by the United States Food and Drug Administration (FDA), and if approved, product launch in the United States is expected in 2020. Remestemcel-L is also being developed for other inflammatory diseases in children and adults including moderate to severe acute respiratory distress syndrome. Mesoblast is completing Phase 3 trials for its product candidates for advanced heart failure and chronic low back pain. Two products have been commercialized in Japan and Europe by Mesoblast’s licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

Forward-Looking Statements This announcement includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. Forward- looking statements include, but are not limited to, statements about: the timing, progress and results of Mesoblast’s preclinical and clinical studies; Mesoblast’s ability to advance product candidates into, enroll and successfully complete, clinical studies; the timing or likelihood of regulatory filings and approvals; and the pricing and reimbursement of Mesoblast’s product candidates, if approved; Mesoblast’s ability to establish and maintain intellectual property on its product candidates and Mesoblast’s ability to successfully defend these in cases of alleged infringement. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblast’s actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. We do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

Release authorized by the Chief Executive.

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Expanded Access Protocol Initiated for Compassionate Use of Remestemcel-L in Children With Multisystem Inflammatory Syndrome Associated With COVID-19...

Quick and Simple Technology Enhances the Potential of Stem Cells To Differentiate Into Adult Cells – Technology Networks

Stem cells have been holding great promise for regenerative medicine for years. In the last decade, several studies have shown that this type of cell, which in Spanish is called mother cell because of its ability to give rise to a variety of different cell types, can be applied in regenerative medicine for diseases such as muscular and nervous system disorders, among others. Researchers and stem cell pioneers Sir John B. Gurdon and Shinya Yamanaka received the Nobel Prize in Physiology and Medicine in 2012 for this idea. However, one of the main limitations in the application of these cell therapies is the quality of the stem cells that can be generated in the laboratory, which impedes their use for therapeutic purposes.Now, a team from the Cell Division and Cancer Group of the Spanish National Cancer Research Centre (CNIO), led by researcher Marcos Malumbres, has developed a new, simple and fast technology that enhances in vitro and in vivo the potential of stem cells to differentiate into adult cells. The research results are published in The EMBO Journal.

In recent years, several protocols have been proposed to obtain reprogrammed stem cells in the laboratory from adult cells, but very few to improve the cells we already have. The method we developed is able to significantly increase the quality of stem cells obtained by any other protocol, thus favouring the efficiency of the production of specialised cell types, says Mara Salazar-Roa, researcher at the CNIO, first author of the article and co-corresponding author.

In this study, the researchers identified an RNA sequence, called microRNA 203, which is found in the earliest embryonic stages before the embryo implants in the womb and when stem cells still have their maximum capacity to generate all the different tissues. When they added this molecule to stem cells in the laboratory, they discovered that the cells ability to convert to other cell types improved significantly.

To corroborate this, they used stem cells of human and murine origin, and of genetically modified mice. The results were spectacular, both in mouse cells and in human cells. Application of this microRNA for just 5 days boosts the potential of stem cells in all scenarios we tested and improves their ability to become other specialised cells, even months after having been in contact with the microRNA, says Salazar-Roa.

According to the study, cells modified by this new protocol are more efficient in generating functional cardiac cells, opening the door to an improved generation of different cell types necessary for the treatment of degenerative diseases.

Malumbres, head of the CNIO Cell and Cancer Division Group, says: To bring this asset to the clinic, collaboration with laboratories or companies that want to exploit this technology is now necessary in each specific case. In this context, Salazar-Roa recently participated, in close collaboration with the CNIOs Innovation team, in prestigious innovation programs such as IDEA2 Global of the Massachusetts Institute of Technology (MIT) and CaixaImpulse of the la Caixa Foundation, from which they also obtained funding to start the development of this technology.

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Quick and Simple Technology Enhances the Potential of Stem Cells To Differentiate Into Adult Cells - Technology Networks

Its not just the lungs: COVID-19 can affect the brain and heart of those infected, researchers say – WITI FOX 6 Milwaukee

LOS ANGELES As medical experts learn about the novel coronavirus, which continues to exhibit an array of ever-evolving symptoms and long-term effects, researchers have found that the deadly illness can have deleterious impacts on the heart and brain.

A recent study published on June 25 in the journalCell Reports Medicine, found that while COVID-19 is commonly known as a respiratory illness, the disease has also been known to instigate inflammatory responses in the body which can negatively affect the function of ones heart and brain.

According to the study, researchers observed SARS-CoV-2 infecting human heart cells that were grown from stem cells in a lab. Within 72 hours of infection, the virus managed to spread and replicate, killing the heart cells.

The researchers brought up the particularly alarming possibility that if COVID-19 can infect the heart cells in a laboratory setting, it could possibly infect those specific organs, prompting the need for a cardiac-specific antiviral drug screen program.

And those concerns are not unwarranted, according to doctors and other researchers who have been observing and studying the wide range of health problems and negative outcomes that appear to come with the not-yet-fully-known territory of the novel virus.

The most common coronavirus symptoms are fever, a dry cough and shortness of breath and some people are contagious despite never experiencing symptoms. But as the virus continues to spread, less common symptoms are being reported, including loss of smell, vomiting and diarrhea, along with a variety of skin problems and harmful neurological effects.

A recentreportfromDr. Robert Stevens, M.D., the associate director of the Johns Hopkins Precision Medicine Center of Excellence for Neurocritical Care, said that coronavirus patients are continuously experiencing a wide range of disconcerting effects on the brain.

Some of the neural symptoms, according to Johns Hopkins, include:

Patients are also having peripheral nerve issues, such as Guillain-Barr syndrome, which can lead to paralysis and respiratory failure, wrote Stevens. I estimate that at least half of the patients Im seeing in the COVID-19 units have neurological symptoms.

While medical experts have continuously repeated that more is still being discovered about the virus, Stevens listed some possibilities on how COVID-19, a respiratory illness, is making its way to the brain.

The first possible way is that the virus may have the capacity to enter the brain and cause a severe and sudden infection. Cases reported in China and Japan found the viruss genetic material in spinal fluid, and a case in Florida found viral particles in brain cells, Stevens wrote.

He added that viral particles in the brain and spine may occur when the virus enters the body through a patients bloodstream or nerve endings.

The second possibility is that the bodys immune system has an overreaction to the virus, causing severe inflammatory responses that cause organ and tissue damage.

The third theory is the erratic physiological changes the disease causes in the body, which involve extremely high fever and low oxygen levels in the blood, result in harmful effects to the brain.

Stevens added that there has been an abnormal observance of blood clotting that has caused some coronavirus patients to suffer strokes. A stroke could occur if a blood clot were to block or narrow arteries leading to the brain, he said.

Another illness that has been known to impact the brain in patients with COVID-19 is currently being studied by Dr. Mady Hornig, an immunologist and professor of epidemiology at Columbia University.

Hornig said that Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an illness that has been found in patients who have recovered from coronaviruses such as SARS.

TheCenters for Disease Control and Preventioncites a 2015 report from the nations top medical advisory body, the Institute of Medicine, which says that an estimated 836,000 to 2.5 million Americans suffer from ME/CFS.

The CDC says that people with ME/CFS experience severe fatigue, sleep problems, as well as difficulty with thinking and concentrating while experiencing pain and dizziness.

Hornig said SARS-CoV-1 and MERS have been associated with longer-term difficulties, in which many people appeared to have symptoms of ME/CFS.

Hornig is currently researching the long-term effects of COVID-19, and has been confronted with an array of concerning symptoms that have persisted in patients, as well as herself.

She can personally attest to the variety of symptoms that have been reported in coronavirus patients, ever since she began to experience her own COVID-19 symptoms in April that have continued to impact her daily life for the past few months.

She has also experienced cardiac complications while dealing with the illness.

Since getting sick, Hornig said shes had to carry a pulse oximeter with her, a device which registers her pulse since she began to have tachycardia episodes when her fever began to decline. Tachycardia is a condition that can make a persons heart beat abnormally fast, reducing blood flow to the rest of the body,according to the Mayo Clinic.

Hornigs most recent episode was on June 22. Her pulse registered at 135 beats per minute, which she said occurred just from her sitting at her computer. She said a normal pulse for someone her age would be around 60-70 beats per minute.

The findings on the novel virus potential effects on the heart and brain come as the CDC continues to update itslistof coronavirus symptoms and high-risk conditions for COVID-19 complications.

Notably, the CDC also removed the specific age threshold from the older adult classification. CDC now warns that among adults, risk increases steadily as you age, and its not just those over the age of 65 who are at increased risk for severe illness, the agency wrote.

Johns Hopkins has noted that younger patients in their 30s and 40s are reportedly having strokes as a result of COVID-19.

It may have something to do with the hyperactive blood-clotting system in these patients, Stevens said. Another system that is hyper-activated in patients with COVID-19 is the endothelial system, which consists of the cells that form the barrier between blood vessels and body tissue. This system is more biologically active in younger patients, and the combination of hyperactive endothelial and blood-clotting systems puts these patients at a major risk for developing blood clots.

But Stevens cautioned that more conclusive data is needed before the medical community can say with assurance that younger people are particularly susceptible to strokes caused by the novel coronavirus.

It is also plausible that theres an increase in stroke in COVID-19 patients of all ages, Stevens said.

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Its not just the lungs: COVID-19 can affect the brain and heart of those infected, researchers say - WITI FOX 6 Milwaukee

Cell Separation Technology Market by Leading Manufacturers, Demand and Growth Overview 2019 to 2027 – Jewish Life News

Transparency Market Research (TMR) has published a new report on the globalcell separation technology marketfor the forecast period of 20192027. According to the report, the global cell separation technology market was valued at ~ US$ 5 Bn in 2018, and is projected to expand at a double-digit CAGR during the forecast period.

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Cell separation, also known as cell sorting or cell isolation, is the process of removing cells from biological samples such as tissue or whole blood. Cell separation is a powerful technology that assists biological research. Rising incidences of chronic illnesses across the globe are likely to boost the development of regenerative medicines or tissue engineering, which further boosts the adoption of cell separation technologies by researchers.

Expansion of the global cell separation technology market is attributed to an increase in technological advancements and surge in investments in research & development, such asstem cellresearch and cancer research. The rising geriatric population is another factor boosting the need for cell separation technologies Moreover, the geriatric population, globally, is more prone to long-term neurological and other chronic illnesses, which, in turn, is driving research to develop treatment for chronic illnesses. Furthermore, increase in the awareness about innovative technologies, such as microfluidics, fluorescent-activated cells sorting, and magnetic activated cells sorting is expected to propel the global cell separation technology market.

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North America dominated the global cell separation technology market in 2018, and the trend is anticipated to continue during the forecast period. This is attributed to technological advancements in offering cell separation solutions, presence of key players, and increased initiatives by governments for advancing the cell separation process. However, insufficient funding for the development of cell separation technologies is likely to hamper the global cell separation technology market during the forecast period. Asia Pacific is expected to be a highly lucrative market for cell separation technology during the forecast period, owing to improving healthcare infrastructure along with rising investments in research & development in the region.

Rising Incidences of Chronic Diseases, Worldwide, Boosting the Demand for Cell Therapy

Incidences of chronic diseases such as diabetes, obesity, arthritis, cardiac diseases, and cancer are increasing due to sedentary lifestyles, aging population, and increased alcohol consumption and cigarette smoking. According to the World Health Organization (WHO), by 2020, the mortality rate from chronic diseases is expected to reach73%, and in developing counties,70%deaths are estimated to be caused by chronic diseases. Southeast Asia, Eastern Mediterranean, and Africa are expected to be greatly affected by chronic diseases. Thus, the increasing burden of chronic diseases around the world is fuelling the demand for cellular therapies to treat chronic diseases. This, in turn, is driving focus and investments on research to develop effective treatments. Thus, increase in cellular research activities is boosting the global cell separation technology market.

Increase in Geriatric Population Boosting the Demand for Surgeries

The geriatric population is likely to suffer from chronic diseases such as cancer and neurological disorders more than the younger population. Moreover, the geriatric population is increasing at a rapid pace as compared to that of the younger population. Increase in the geriatric population aged above 65 years is projected to drive the incidences of Alzheimers, dementia, cancer, and immune diseases, which, in turn, is anticipated to boost the need for corrective treatment of these disorders. This is estimated to further drive the demand for clinical trials and research that require cell separation products. These factors are likely to boost the global cell separation technology market.

According to the United Nations, the geriatric population aged above 60 is expected to double by 2050 and triple by 2100, an increase from962 millionin 2017 to2.1 billionin 2050 and3.1 billionby 2100.

Productive Partnerships in Microfluidics Likely to Boost the Cell Separation Technology Market

Technological advancements are prompting companies to innovate in microfluidics cell separation technology. Strategic partnerships and collaborations is an ongoing trend, which is boosting the innovation and development of microfluidics-based products. Governments and stakeholders look upon the potential in single cell separation technology and its analysis, which drives them to invest in the development ofmicrofluidics. Companies are striving to build a platform by utilizing their expertise and experience to further offer enhanced solutions to end users.

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Stem Cell Research to Account for a Prominent Share

Stem cell is a prominent cell therapy utilized in the development of regenerative medicine, which is employed in the replacement of tissues or organs, rather than treating them. Thus, stem cell accounted for a prominent share of the global market. The geriatric population is likely to increase at a rapid pace as compared to the adult population, by 2030, which is likely to attract the use of stem cell therapy for treatment. Stem cells require considerably higher number of clinical trials, which is likely to drive the demand for cell separation technology, globally. Rising stem cell research is likely to attract government and private funding, which, in turn, is estimated to offer significant opportunity for stem cell therapies.

Biotechnology & Pharmaceuticals Companies to Dominate the Market

The number of biotechnology companies operating across the globe is rising, especially in developing countries. Pharmaceutical companies are likely to use cells separation techniques to develop drugs and continue contributing through innovation. Growing research in stem cell has prompted companies to own large separate units to boost the same. Thus, advancements in developing drugs and treatments, such as CAR-T through cell separation technologies, are likely to drive the segment.

As per research, 449 public biotech companies operate in the U.S., which is expected to boost the biotechnology & pharmaceutical companies segment. In developing countries such as China, China Food and Drug Administration (CFDA) reforms pave the way for innovation to further boost biotechnology & pharmaceutical companies in the country.

Global Cell Separation Technology Market: Prominent Regions

North America to Dominate Global Market, While Asia Pacific to Offer Significant Opportunity

In terms of region, the global cell separation technology market has been segmented into five major regions: North America, Europe, Asia Pacific, Latin America, and the Middle East & Africa. North America dominated the global market in 2018, followed by Europe. North America accounted for a major share of the global cell separation technology market in 2018, owing to the development of cell separation advanced technologies, well-defined regulatory framework, and initiatives by governments in the region to further encourage the research industry. The U.S. is a major investor in stem cell research, which accelerates the development of regenerative medicines for the treatment of various long-term illnesses.

The cell separation technology market in Asia Pacific is projected to expand at a high CAGR from 2019 to 2027. This can be attributed to an increase in healthcare expenditure and large patient population, especially in countries such as India and China. Rising medical tourism in the region and technological advancements are likely to drive the cell separation technology market in the region.

Launching Innovative Products, and Acquisitions & Collaborations by Key Players Driving Global Cell Separation Technology Market

The global cell separation technology market is highly competitive in terms of number of players. Key players operating in the global cell separation technology market include Akadeum Life Sciences, STEMCELL Technologies, Inc., BD, Bio-Rad Laboratories, Inc., Miltenyi Biotech, 10X Genomics, Thermo Fisher Scientific, Inc., Zeiss, GE Healthcare Life Sciences, PerkinElmer, Inc., and QIAGEN.

These players have adopted various strategies such as expanding their product portfolios by launching new cell separation kits and devices, and participation in acquisitions, establishing strong distribution networks. Companies are expanding their geographic presence in order sustain in the global cell separation technology market. For instance, in May 2019, Akadeum Life Sciences launched seven new microbubble-based products at a conference. In July 2017, BD received the U.S. FDAs clearance for its BD FACS Lyric flow cytometer system, which is used in the diagnosis of immunological disorders.

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Cell Separation Technology Market by Leading Manufacturers, Demand and Growth Overview 2019 to 2027 - Jewish Life News

Interim Analysis of Recardio’s Phase II Clinical Trial to Be Presented at the 2020 Congress of the European Society of Cardiology – PRNewswire

SAN FRANCISCO, June 29, 2020 /PRNewswire/ --Entitled "Randomized, Double Blind, Placebo-Controlled, Safety and Efficacy Study of Dutogliptin in Combination with Filgrastim in Early Recovery Post-Myocardial Infarction: rationale, design and first interim analysis", the presentation provides an initial insight into patient outcomes of the trial that is currently ongoing in multiple centers. Patients included in this trial experienced a severe form of Myocardial Infarction known as STEMI. Soon after the initial intervention to re-establish adequate blood flow to the coronary arteries, patients begin a two-week treatment with Recardio's dutogliptin, a small molecule that enables sustained homing of mobilised stem cells to the site of cardiac injury. By releasing paracrine factors, stem cells have been shown to have significant repair and regenerative effects that improve healing and recovery of cardiac function after the infarction.

More information about the clinical program is available by visiting the "clinicaltrials.gov" website at the following link:https://clinicaltrials.gov/ct2/show/NCT03486080

About Recardio

Recardio Inc. is a clinical-stage life science company focusing ontherapies for cardiovascular, oncology and infectious diseases. The company is located in San Francisco, California, and hasoperations in the USA and Europe.The company's lead drug candidate, dutogliptin, is a DPP-IV inhibitor that has demonstrated significant effects in activating various chemokines like SDF-1, a protein that is critical for cardiac regeneration. In addition to its current Phase 2 cardiovascular clinical program, Recardio will fully develop the therapeutic platform as a regenerative medication for patients with various cardiovascular diseases including acute myocardial infarction and chronic heart failure, with the potential of improving heart function, quality of life and survival.

For more information, visit:http://www.recardio.eu/or contact[emailprotected]

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Interim Analysis of Recardio's Phase II Clinical Trial to Be Presented at the 2020 Congress of the European Society of Cardiology - PRNewswire

Exosome Therapeutic Market Size, 2020-New Technological Change Helping Market, Application, Driver, – PharmiWeb.com

Pune, Maharashtra, India, June 29 2020 (Wiredrelease) Data Bridge Market Research A New Business Intelligence Report released by Data Bridge Market Research with title Global Exosome Therapeutic Market size, share, growth, Industry Trends and Forecast 2027 has abilities to raise as the most significant market worldwide as it has remained playing a remarkable role in establishing progressive impacts on the universal economy. The Global Exosome Therapeutic Market Report offers energetic visions to conclude and study the market size, market hopes, and competitive surroundings. The research is derived through primary and secondary statistics sources and it comprises both qualitative and quantitative detailing. This report has been crafted as the result of persistent efforts lead by knowledgeable forecasters, innovative analysts and brilliant researchers who indulge in detailed and diligent research on different markets, trends and emerging opportunities in the successive direction for the business needs.

Download Exclusive Sample Report (350 Pages PDF with All Related Graphs & Charts) For Free@:https://www.databridgemarketresearch.com/request-a-sample/?dbmr=global-exosome-therapeutic-market

DBMR Analyses that the Exosome Therapeutic Market is growing with a CAGR of 21.9% in the forecast period of 2019 to 2026 and expected to reach USD 31,691.52 million by 2026 from USD 6,500.00 million in 2018. Increasing prevalence of lyme disease, chronic inflammation, autoimmune disease and other chronic degenerative diseases are the factors for the market growth.

Increased number of exosome therapeutics as compared to the past few years will accelerate market growth. Companies are receiving funding for exosome therapeutic research and clinical trials. For instance, In September 2018, EXOCOBIO has raised USD 27 million in its series B funding. The company has raised USD 46 million as series a funding in April 2017. The series B funding will help the company to set up GMP-compliant exosome industrial facilities to enhance production of exosomes to commercialize in cosmetics and pharmaceutical industry.

KNOW YOUR OPTIONS IN THE FIGHT AGAINST COVID-19

The COVID-19 Pandemic has created bottlenecks across industry pipelines, sales funnels, and supply chain activities. This has created unprecedented budget pressure on company spending for industry leaders. This has increased requirement for opportunity analysis, price trend knowledge and competitive outcomes. Use the DBMR team to create new sales channels and capture new markets previously unknown. DBMR helps its clients to grow in these uncertain markets.

To Understand How COVID-19 Impact is covered in This Report. Get Sample Copy of the Report@https://www.databridgemarketresearch.com/request-covid-19/global-exosome-therapeutic-market

The Global Exosome Therapeutic market 2020 research provides a basic overview of the industry including definitions, classifications, applications and industry chain structure. The Global Exosome Therapeutic Market Share analysis is provided for the international markets including development trends, competitive landscape analysis, and key regions development status. Development policies and plans are discussed as well as manufacturing processes and cost structures are also analyzed. This report also states import/export consumption, supply and demand Figures, cost, price, revenue and gross margins. For each manufacturer covered, this report analyzes their Exosome Therapeutic manufacturing sites, capacity, production, ex-factory price, revenue and market share in global market.

Major Players in Global Exosome Therapeutic Market Include

evox THERAPEUTICSEXOCOBIOExopharmAEGLE TherapeuticsUnited Therapeutics CorporationCodiak BioSciencesJazz Pharmaceuticals, Inc.Boehringer Ingelheim International GmbHReNeuron Group plcCapricor TherapeuticsAvalon Globocare Corp.CREATIVE MEDICAL TECHNOLOGY HOLDINGS INC.Stem Cells Group..

Complete Report is Available (Including Full TOC, List of Tables & Figures, Graphs, and Chart)@https://www.databridgemarketresearch.com/toc/?dbmr=global-exosome-therapeutic-market

New Exosome Therapeutic Market Developments in 2019

In January 2019, Codiak BioSciences has collaborated with Jazz Pharmaceuticals, Inc. to develop and commercialize exosome therapeutics to treat cancer. The collaboration will help the company to address issues which have been often implicated in solid tumors and hematological malignancies.

In October 2018, Avalon GloboCare Corp. has collaborated with Weill Cornell Medicine to form standards in cGMP-grade for human endothelial cells sourced exosome which is significant for organ regeneration and vascular health and isolation and identification of exosomes sourced from tissue for liquid biopsy and clinical use. The collaboration will help the company to lead market as exosome isolation system as will be first in the world for standardization processing of cGMP-grade exosomes for clinical studies.

In July 2018, Capricor Therapeutics has formed collaboration with the U.S. Army Institute of Surgical Research (USAISR) to discover potential for CAP-2003 (exosomes) in order to address trauma-related conditions and injuries. The collaboration will help to test CAP-2003 as a tool for preservation of life.

This research is categorized differently considering the various aspects of this market. It also evaluates the current situation and the future of the market by using the forecast horizon. The forecast is analyzed based on the volume and revenue of this market. The tools used for analyzing the Global Exosome Therapeutic Market research report include SWOT analysis.

The Global Exosome Therapeutic segments and Market Data Break Down are illuminated below:

By Type (Natural Exosomes, Hybrid Exosomes

By Source (Dendritic Cells, Mesenchymal Stem Cells, Blood, Milk, Body Fluids, Saliva, Urine Others)

By Therapy (Immunotherapy, Gene Therapy, Chemotherapy)

By Transporting Capacity (Bio Macromolecules, Small Molecules

By Application (Oncology, Neurology, Metabolic Disorders, Cardiac Disorders, Blood Disorders, Inflammatory Disorders, Gynecology Disorders, Organ Transplantation, Others)

By Route of administration (Oral, Parenteral)

By End User (Hospitals, Diagnostic Centers, Research & Academic Institutes)

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The Global Exosome Therapeutic Market in terms of investment potential in various segments of the market and illustrate the feasibility of explaining the feasibility of a new project to be successful in the near future. The core segmentation of the global market is based on product types, SMEs and large corporations. The report also collects data for each major player in the market based on current company profiles, gross margins, sales prices, sales revenue, sales volume, photos, product specifications and up-to-date contact information.

What are the strengths and weaknesses of the key vendors?

Definitively, this report will give you an unmistakable perspective on every single reality of the market without a need to allude to some other research report or an information source. Our report will give all of you the realities about the past, present, and eventual fate of the concerned Market.

Scope of the Exosome Therapeutic Market

The global exosome therapeutic market is segmented on the basis of countries into U.S., Mexico, Turkey, Hong Kong, Australia, South Korea, Argentina, Colombia, Peru, Chile, Ecuador, Venezuela, Panama, Dominican Republic, El Salvador, Paraguay, Costa Rica, Puerto Rico, Nicaragua and Uruguay.

All country based analysis of the exosome therapeutic market is further analyzed based on maximum granularity into further segmentation. On the basis of type, the market is segmented into natural exosomes and hybrid exosomes. Based on source, the market is segmented into dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, urine and others. On the basis of therapy, the market is segmented into immunotherapy, gene therapy and chemotherapy. On the basis of transporting capacity, the market is segmented into bio macromolecules and small molecules. On the basis of application, the market is segmented into oncology, neurology, metabolic disorders, cardiac disorders, blood disorders, inflammatory disorders, gynecology disorders, organ transplantation and others. On the basis of route of administration, the market is segmented into pa oral and parenteral. On the basis of end user, the market is segmented into hospitals, diagnostic centers and research & academic institutes and others.

Some Points from Table of Content:

1 Report Overview1.1 Study Scope1.2 Key Market Segments1.3 Regulatory Scenario by Region/Country1.4 Market Investment Scenario Strategic1.5 Market Analysis by Type1.5.1 Global Exosome Therapeutic Market Share by Type (2020-2027)1.5.2 Type 11.5.3 Type 21.5.4 Other1.6 Market by Application1.6.1 Global Exosome Therapeutic Market Share by Application (2020-2027)1.6.2 Application 11.6.3 Application 21.6.4 Other1.7 Exosome Therapeutic Industry Development Trends under COVID-19 Outbreak1.7.1 Region COVID-19 Status Overview1.7.2 Influence of COVID-19 Outbreak on Exosome Therapeutic Industry Development

Global Market Growth Trends2.1 Industry Trends2.1.1 SWOT Analysis2.1.2 Porters Five Forces Analysis2.2 Potential Market and Growth Potential Analysis2.3 Industry News and Policies by Regions2.3.1 Industry News2.3.2 Industry Policies3 Value Chain of Exosome Therapeutic Market3.1 Value Chain Status3.2 Exosome Therapeutic Manufacturing Cost Structure Analysis3.2.1 Production Process Analysis3.2.2 Manufacturing Cost Structure of Exosome Therapeutic3.2.3 Labor Cost of Exosome Therapeutic3.3 Sales and Marketing Model Analysis3.4 Downstream Major Customer Analysis (by Region)

4 Players Profiles4.1 Player 14.1.1 Player 1 Basic Information4.1.2 Exosome Therapeutic Product Profiles, Application and Specification4.1.3 Player 1 Exosome Therapeutic Market Performance (2015-2020)4.1.4 Player 1 Business Overview4.2 Player 24.2.1 Player 2 Basic Information4.2.2 Exosome Therapeutic Product Profiles, Application and Specification4.2.3 Player 2 Exosome Therapeutic Market Performance (2015-2020)4.2.4 Player 2 Business Overview4.3 Player 34.3.1 Player 3 Basic Information4.3.2 Exosome Therapeutic Product Profiles, Application and Specification4.3.3 Player 3 Exosome Therapeutic Market Performance (2015-2020)4.3.4 Player 3 Business Overview4.4 Player 44.4.1 Player 4 Basic Information4.4.2 Exosome Therapeutic Product Profiles, Application and Specification4.4.3 Player 4 Exosome Therapeutic Market Performance (2015-2020)4.4.4 Player 4 Business Overview4.5 Player 54.5.1 Player 5 Basic Information4.5.2 Exosome Therapeutic Product Profiles, Application and Specification4.5.3 Player 5 Exosome Therapeutic Market Performance (2015-2020)

4.5.4 Player 5 Business Overview5 Global Exosome Therapeutic Market Analysis by Regions5.1 Global Exosome Therapeutic Sales, Revenue and Market Share by Regions5.1.1 Global Exosome Therapeutic Sales by Regions (2015-2020)5.1.2 Global Exosome Therapeutic Revenue by Regions (2015-2020)5.2 North America Exosome Therapeutic Sales and Growth Rate (2015-2020)5.3 Europe Exosome Therapeutic Sales and Growth Rate (2015-2020)5.4 Asia-Pacific Exosome Therapeutic Sales and Growth Rate (2015-2020)5.5 Middle East and Africa Exosome Therapeutic Sales and Growth Rate (2015-2020)5.6 South America Exosome Therapeutic Sales and Growth Rate (2015-2020)

11 Global Exosome Therapeutic Market Segment byTypes12 Global Exosome Therapeutic Market Segment by Applications13 Exosome Therapeutic Market Forecast by Regions (2020-2027)ContinuedComplete Report Is Available| Get Free TOC @https://www.databridgemarketresearch.com/toc/?dbmr=global-exosome-therapeutic-market

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Exosome Therapeutic Market Size, 2020-New Technological Change Helping Market, Application, Driver, - PharmiWeb.com

FDA Approves Merck’s KEYTRUDA (pembrolizumab) for First-Line Treatment of Patients With Unresectable or Metastatic MSI-H or dMMR Colorectal Cancer -…

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the U.S. Food and Drug Administration (FDA) has approved KEYTRUDA, Mercks anti-PD-1 therapy, as monotherapy for the first-line treatment of patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) colorectal cancer. The approval is based on results from the Phase 3 KEYNOTE-177 trial, in which KEYTRUDA significantly reduced the risk of disease progression or death by 40% (HR=0.60 [95% CI, 0.45-0.80; p=0.0004]) compared with chemotherapy, the current standard of care. In the study, treatment with KEYTRUDA also more than doubled median progression-free survival (PFS) compared with chemotherapy (16.5 months [95% CI, 5.4-32.4] versus 8.2 months [95% CI, 6.1-10.2]).

Todays approval has the potential to change the treatment paradigm for the first-line treatment of patients with MSI-H colorectal cancer, based on the important findings from KEYNOTE-177 that showed KEYTRUDA monotherapy demonstrated superior progression-free survival compared to standard of care chemotherapy, said Dr. Roy Baynes, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. Our commitment to pursuing biomarker research continues to help us bring new treatments to patients, particularly for those who have few available options.

Immune-mediated adverse reactions, which may be severe or fatal, can occur with KEYTRUDA, including pneumonitis, colitis, hepatitis, endocrinopathies, nephritis and renal dysfunction, severe skin reactions, solid organ transplant rejection, and complications of allogeneic hematopoietic stem cell transplantation (HSCT). Based on the severity of the adverse reaction, KEYTRUDA should be withheld or discontinued and corticosteroids administered if appropriate. KEYTRUDA can also cause severe or life-threatening infusion-related reactions. Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. For more information, see Selected Important Safety Information below.

This approval was granted less than one month following the submission of a new supplemental Biologics License Application (sBLA), which was reviewed under the FDAs Real-Time Oncology Review (RTOR) pilot program. This review also was conducted under Project Orbis, an initiative of the FDA Oncology Center of Excellence that provides a framework for concurrent submission and review of oncology drugs among its international partners. For this application, a modified Project Orbis was undertaken, and the FDA is collaborating with the Australian Therapeutic Goods Administration, Health Canada and Swissmedic on their ongoing review of the application.

Patients with unresectable or metastatic MSI-H colorectal cancer have historically faced poor outcomes, and until today, chemotherapy-containing regimens were the only FDA-approved first-line treatment options, said Luis A. Diaz, M.D., head of the division of Solid Tumor Oncology, Memorial Sloan Kettering Cancer Center. In patients who were treated with KEYTRUDA and responded (n=67) in the KEYNOTE-177 trial, 43% of patients experienced a duration of response lasting two years or longer. This approval helps address the unmet need to provide a new monotherapy treatment option for patients.

Data Supporting the Approval

The approval was based on data from KEYNOTE-177 (NCT02563002), a multi-center, randomized, open-label, active-controlled trial that enrolled 307 patients with previously untreated unresectable or metastatic MSI-H or dMMR colorectal cancer. Microsatellite instability (MSI) or mismatch repair (MMR) tumor status was determined locally using polymerase chain reaction or immunohistochemistry, respectively. Patients with autoimmune disease or a medical condition that required immunosuppression were ineligible.

Patients were randomized 1:1 to receive KEYTRUDA 200 mg intravenously every three weeks or investigators choice of the following chemotherapy regimens given intravenously every two weeks:

Treatment with KEYTRUDA or chemotherapy continued until Response Evaluation Criteria in Solid Tumors (RECIST) v1.1-defined progression of disease as determined by the investigator or unacceptable toxicity. Patients treated with KEYTRUDA without disease progression could be treated for up to 24 months. Assessment of tumor status was performed every nine weeks. Patients randomized to chemotherapy were offered KEYTRUDA at the time of disease progression. The main efficacy outcome measure was progression-free survival (PFS) as assessed by blinded independent central review (BICR) according to RECIST v1.1, modified to follow a maximum of 10 target lesions and a maximum of five target lesions per organ, and overall survival (OS). Additional efficacy outcome measures were objective response rate (ORR) and duration of response (DOR).

Patients were enrolled and randomized to KEYTRUDA (n=153) or chemotherapy (n=154). The baseline characteristics of these 307 patients were: median age of 63 years (range, 24 to 93), 47% age 65 or older; 50% male; 75% White and 16% Asian; 52% had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0, and 48% had an ECOG PS of 1; and 27% received prior adjuvant or neoadjuvant chemotherapy. Among the 154 patients randomized to receive chemotherapy, 143 received chemotherapy per the protocol. Of these 143 patients, 56% received mFOLFOX6, 44% received FOLFIRI, 70% received bevacizumab plus mFOLFOX6 or FOLFIRI, and 11% received cetuximab plus mFOLFOX6 or FOLFIRI. The median follow-up time was 27.6 months (range, 0.2 to 48.3 months).

In this study, KEYTRUDA monotherapy significantly reduced the risk of disease progression or death by 40% (HR=0.60 [95% CI, 0.45-0.80; p=0.0004]) and showed a median PFS of 16.5 months (95% CI, 5.4-32.4) compared with 8.2 months (95% CI, 6.1-10.2) for patients treated with chemotherapy. For PFS, in the KEYTRUDA arm, there were 82 patients (54%) with an event versus 113 patients (73%) in the chemotherapy arm. At the time of the PFS analysis, the OS data were not mature (66% of the required number of events for the OS final analysis). For patients treated with KEYTRUDA, the ORR was 44% (95% CI, 35.8-52.0), with a complete response rate of 11% and a partial response rate of 33%, and for patients treated with chemotherapy, the ORR was 33% (95% CI, 25.8-41.1), with a complete response rate of 4% and a partial response rate of 29%. Median DOR was not reached (range, 2.3+ to 41.4+) with KEYTRUDA versus 10.6 months (range, 2.8 to 37.5+) with chemotherapy. Based on 67 patients with a response in the KEYTRUDA arm and 51 patients with a response in the chemotherapy arm, 75% in the KEYTRUDA arm had a duration of response greater than or equal to 12 months versus 37% in the chemotherapy arm, and 43% in the KEYTRUDA arm had a duration of response greater than or equal to 24 months versus 18% in the chemotherapy arm.

Among the 153 patients with MSI-H or dMMR colorectal cancer treated with KEYTRUDA, the median duration of exposure to KEYTRUDA was 11.1 months (range, 1 day to 30.6 months). Adverse reactions occurring in patients with MSI-H or dMMR colorectal cancer were similar to those occurring in 2,799 patients with melanoma or non-small cell lung cancer treated with KEYTRUDA as a single agent.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,200 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory classical Hodgkin lymphoma (cHL), or who have relapsed after 3 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 [combined positive score (CPS) 10], as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High (MSI-H) Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High (TMB-H)

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase (mut/Mb)] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Selected Important Safety Information for KEYTRUDA

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis, including fatal cases. Pneumonitis occurred in 3.4% (94/2799) of patients with various cancers receiving KEYTRUDA, including Grade 1 (0.8%), 2 (1.3%), 3 (0.9%), 4 (0.3%), and 5 (0.1%). Pneumonitis occurred in 8.2% (65/790) of NSCLC patients receiving KEYTRUDA as a single agent, including Grades 3-4 in 3.2% of patients, and occurred more frequently in patients with a history of prior thoracic radiation (17%) compared to those without (7.7%). Pneumonitis occurred in 6% (18/300) of HNSCC patients receiving KEYTRUDA as a single agent, including Grades 3-5 in 1.6% of patients, and occurred in 5.4% (15/276) of patients receiving KEYTRUDA in combination with platinum and FU as first-line therapy for advanced disease, including Grades 3-5 in 1.5% of patients.

Monitor patients for signs and symptoms of pneumonitis. Evaluate suspected pneumonitis with radiographic imaging. Administer corticosteroids for Grade 2 or greater pneumonitis. Withhold KEYTRUDA for Grade 2; permanently discontinue KEYTRUDA for Grade 3 or 4 or recurrent Grade 2 pneumonitis.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis. Colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 2 (0.4%), 3 (1.1%), and 4 (<0.1%). Monitor patients for signs and symptoms of colitis. Administer corticosteroids for Grade 2 or greater colitis. Withhold KEYTRUDA for Grade 2 or 3; permanently discontinue KEYTRUDA for Grade 4 colitis.

Immune-Mediated Hepatitis (KEYTRUDA) and Hepatotoxicity (KEYTRUDA in Combination With Axitinib)

Immune-Mediated Hepatitis

KEYTRUDA can cause immune-mediated hepatitis. Hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.4%), and 4 (<0.1%). Monitor patients for changes in liver function. Administer corticosteroids for Grade 2 or greater hepatitis and, based on severity of liver enzyme elevations, withhold or discontinue KEYTRUDA.

Hepatotoxicity in Combination With Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity with higher than expected frequencies of Grades 3 and 4 ALT and AST elevations compared to KEYTRUDA alone. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased ALT (20%) and increased AST (13%) were seen. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider more frequent monitoring of liver enzymes as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed.

Immune-Mediated Endocrinopathies

KEYTRUDA can cause adrenal insufficiency (primary and secondary), hypophysitis, thyroid disorders, and type 1 diabetes mellitus. Adrenal insufficiency occurred in 0.8% (22/2799) of patients, including Grade 2 (0.3%), 3 (0.3%), and 4 (<0.1%). Hypophysitis occurred in 0.6% (17/2799) of patients, including Grade 2 (0.2%), 3 (0.3%), and 4 (<0.1%). Hypothyroidism occurred in 8.5% (237/2799) of patients, including Grade 2 (6.2%) and 3 (0.1%). The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC (16%) receiving KEYTRUDA, as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. Hyperthyroidism occurred in 3.4% (96/2799) of patients, including Grade 2 (0.8%) and 3 (0.1%), and thyroiditis occurred in 0.6% (16/2799) of patients, including Grade 2 (0.3%). Type 1 diabetes mellitus, including diabetic ketoacidosis, occurred in 0.2% (6/2799) of patients.

Monitor patients for signs and symptoms of adrenal insufficiency, hypophysitis (including hypopituitarism), thyroid function (prior to and periodically during treatment), and hyperglycemia. For adrenal insufficiency or hypophysitis, administer corticosteroids and hormone replacement as clinically indicated. Withhold KEYTRUDA for Grade 2 adrenal insufficiency or hypophysitis and withhold or discontinue KEYTRUDA for Grade 3 or Grade 4 adrenal insufficiency or hypophysitis. Administer hormone replacement for hypothyroidism and manage hyperthyroidism with thionamides and beta-blockers as appropriate. Withhold or discontinue KEYTRUDA for Grade 3 or 4 hyperthyroidism. Administer insulin for type 1 diabetes, and withhold KEYTRUDA and administer antihyperglycemics in patients with severe hyperglycemia.

Immune-Mediated Nephritis and Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.1%), and 4 (<0.1%) nephritis. Nephritis occurred in 1.7% (7/405) of patients receiving KEYTRUDA in combination with pemetrexed and platinum chemotherapy. Monitor patients for changes in renal function. Administer corticosteroids for Grade 2 or greater nephritis. Withhold KEYTRUDA for Grade 2; permanently discontinue for Grade 3 or 4 nephritis.

Immune-Mediated Skin Reactions

Immune-mediated rashes, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) (some cases with fatal outcome), exfoliative dermatitis, and bullous pemphigoid, can occur. Monitor patients for suspected severe skin reactions and based on the severity of the adverse reaction, withhold or permanently discontinue KEYTRUDA and administer corticosteroids. For signs or symptoms of SJS or TEN, withhold KEYTRUDA and refer the patient for specialized care for assessment and treatment. If SJS or TEN is confirmed, permanently discontinue KEYTRUDA.

Other Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue in patients receiving KEYTRUDA and may also occur after discontinuation of treatment. For suspected immune-mediated adverse reactions, ensure adequate evaluation to confirm etiology or exclude other causes. Based on the severity of the adverse reaction, withhold KEYTRUDA and administer corticosteroids. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Based on limited data from clinical studies in patients whose immune-related adverse reactions could not be controlled with corticosteroid use, administration of other systemic immunosuppressants can be considered. Resume KEYTRUDA when the adverse reaction remains at Grade 1 or less following corticosteroid taper. Permanently discontinue KEYTRUDA for any Grade 3 immune-mediated adverse reaction that recurs and for any life-threatening immune-mediated adverse reaction.

The following clinically significant immune-mediated adverse reactions occurred in less than 1% (unless otherwise indicated) of 2799 patients: arthritis (1.5%), uveitis, myositis, Guillain-Barr syndrome, myasthenia gravis, vasculitis, pancreatitis, hemolytic anemia, sarcoidosis, and encephalitis. In addition, myelitis and myocarditis were reported in other clinical trials, including classical Hodgkin lymphoma, and postmarketing use.

Treatment with KEYTRUDA may increase the risk of rejection in solid organ transplant recipients. Consider the benefit of treatment vs the risk of possible organ rejection in these patients.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% (6/2799) of patients. Monitor patients for signs and symptoms of infusion-related reactions. For Grade 3 or 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Immune-mediated complications, including fatal events, occurred in patients who underwent allogeneic HSCT after treatment with KEYTRUDA. Of 23 patients with cHL who proceeded to allogeneic HSCT after KEYTRUDA, 6 (26%) developed graft-versus-host disease (GVHD) (1 fatal case) and 2 (9%) developed severe hepatic veno-occlusive disease (VOD) after reduced-intensity conditioning (1 fatal case). Cases of fatal hyperacute GVHD after allogeneic HSCT have also been reported in patients with lymphoma who received a PD-1 receptorblocking antibody before transplantation. Follow patients closely for early evidence of transplant-related complications such as hyperacute graft-versus-host disease (GVHD), Grade 3 to 4 acute GVHD, steroid-requiring febrile syndrome, hepatic veno-occlusive disease (VOD), and other immune-mediated adverse reactions.

In patients with a history of allogeneic HSCT, acute GVHD (including fatal GVHD) has been reported after treatment with KEYTRUDA. Patients who experienced GVHD after their transplant procedure may be at increased risk for GVHD after KEYTRUDA. Consider the benefit of KEYTRUDA vs the risk of GVHD in these patients.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with a PD-1 or PD-L1 blocking antibody in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-002, KEYTRUDA was permanently discontinued due to adverse reactions in 12% of 357 patients with advanced melanoma; the most common (1%) were general physical health deterioration (1%), asthenia (1%), dyspnea (1%), pneumonitis (1%), and generalized edema (1%). The most common adverse reactions were fatigue (43%), pruritus (28%), rash (24%), constipation (22%), nausea (22%), diarrhea (20%), and decreased appetite (20%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

In KEYNOTE-189, when KEYTRUDA was administered with pemetrexed and platinum chemotherapy in metastatic nonsquamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 20% of 405 patients. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonitis (3%) and acute kidney injury (2%). The most common adverse reactions (20%) with KEYTRUDA were nausea (56%), fatigue (56%), constipation (35%), diarrhea (31%), decreased appetite (28%), rash (25%), vomiting (24%), cough (21%), dyspnea (21%), and pyrexia (20%).

In KEYNOTE-407, when KEYTRUDA was administered with carboplatin and either paclitaxel or paclitaxel protein-bound in metastatic squamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 15% of 101 patients. The most frequent serious adverse reactions reported in at least 2% of patients were febrile neutropenia, pneumonia, and urinary tract infection. Adverse reactions observed in KEYNOTE-407 were similar to those observed in KEYNOTE-189 with the exception that increased incidences of alopecia (47% vs 36%) and peripheral neuropathy (31% vs 25%) were observed in the KEYTRUDA and chemotherapy arm compared to the placebo and chemotherapy arm in KEYNOTE-407.

In KEYNOTE-042, KEYTRUDA was discontinued due to adverse reactions in 19% of 636 patients with advanced NSCLC; the most common were pneumonitis (3%), death due to unknown cause (1.6%), and pneumonia (1.4%). The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia (7%), pneumonitis (3.9%), pulmonary embolism (2.4%), and pleural effusion (2.2%). The most common adverse reaction (20%) was fatigue (25%).

In KEYNOTE-010, KEYTRUDA monotherapy was discontinued due to adverse reactions in 8% of 682 patients with metastatic NSCLC; the most common was pneumonitis (1.8%). The most common adverse reactions (20%) were decreased appetite (25%), fatigue (25%), dyspnea (23%), and nausea (20%).

Adverse reactions occurring in patients with SCLC were similar to those occurring in patients with other solid tumors who received KEYTRUDA as a single agent.

In KEYNOTE-048, KEYTRUDA monotherapy was discontinued due to adverse events in 12% of 300 patients with HNSCC; the most common adverse reactions leading to permanent discontinuation were sepsis (1.7%) and pneumonia (1.3%). The most common adverse reactions (20%) were fatigue (33%), constipation (20%), and rash (20%).

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FDA Approves Merck's KEYTRUDA (pembrolizumab) for First-Line Treatment of Patients With Unresectable or Metastatic MSI-H or dMMR Colorectal Cancer -...

Rahul Gandhi to interact with nurses on July 1 – WeForNews

New York, July 1 : A team of US scientists, led by an Indian-origin researcher revealed that SARS-CoV-2 (coronavirus), the virus behind Covid-19, can infect heart cells in a lab dish.

This suggests it may be possible for heart cells in Covid-19 patients to be directly infected by the virus.

The discovery, published today in the journal Cell Reports Medicine, was made using heart muscle cells that were produced by stem cell technology.

We not only uncovered that these stem cell-derived heart cells are susceptible to infection by a novel coronavirus, but that the virus can also quickly divide within the heart muscle cells, said study researcher Arun Sharma from the Cedars-Sinai Board of Governors Regenerative Medicine Institute in the US.

Even more significant, the infected heart cells showed changes in their ability to beat after 72 hours of infection, Sharma added.Although many COVID-19 patients experience heart problems, the reasons remain unclear. Pre-existing cardiac conditions or inflammation and oxygen deprivation resulting from the infection have all been implicated.

But there has until now been only limited evidence the SARS-CoV-2 virus directly infects the individual muscle cells of the heart.The study also demonstrated human stem cell-derived heart cells infected by SARS-CoV-2 change their gene expression profile.This offers further confirmation the cells can be actively infected by the virus and activate innate cellular defence mechanisms in an effort to help clear-out the virus.

This viral pandemic is predominately defined by respiratory symptoms, but there are also cardiac complications, including arrhythmia, heart failure and viral myocarditis, said study co-author Clive Svendsen.

While this could be the result of massive inflammation in response to the virus, our data suggest that the heart could also be directly affected by the virus in Covid-19, Svendsen added.

Researchers also found that treatment with an ACE2 antibody was able to blunt viral replication on stem cell-derived heart cells, suggesting that the ACE2 receptor could be used by SARS-CoV-2 to enter human heart muscle cells.

By blocking the ACE2 protein with an antibody, the virus is not as easily able to bind to the ACE2 protein, and thus cannot easily enter the cell, said Sharma. This not only helps us understand the mechanisms of how this virus functions, but also suggests therapeutic approaches that could be used as a potential treatment for SARS-CoV-2 infection, he explained.

The study used human induced pluripotent stem cells (iPSCs), a type of stem cell that is created in the lab from a persons blood or skin cells. IPSCs can make any cell type found in the body, each one carrying the DNA of the individual. This work illustrates the power of being able to study human tissue in a dish, the authors wrote.

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Rahul Gandhi to interact with nurses on July 1 - WeForNews

Latest News 2020: Autologous Stem Cell Based Therapies Market by Coronavirus-COVID19 Impact Analysis With Top Manufacturers Analysis | Top Players:…

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4D physiologically adaptable cardiac patch: A 4-month in vivo study for the treatment of myocardial infarction – Science Advances

INTRODUCTION

Cardiovascular disease associated with myocardial infarction (MI) is a major cause of morbidity and mortality worldwide (1, 2). The heart is composed of dynamic and multicellular tissues that exhibit highly specific structural and functional characteristics. Adult cardiac muscle is thought to lack the ability to self-repair and regenerate after MI. Traditional cardiac patches serve as temporary mechanical supporting systems to prevent the progression of postinfarction left ventricular (LV) remodeling (2). However, the damaged myocardium is still unable to self-restore, and the subsequent maladaptive remodeling is typically irreversible (2). Because of the shortage of organ donors and the limited retention of cellular therapies, the field of cardiac engineering has emerged to generate functional cardiac tissues to provide a promising alternative means to repair damaged heart tissue (3, 4). In addition to playing a role in providing mechanical support, cellularized cardiac patches and scaffolds have also been investigated to restore the functionality of the damaged myocardium (5, 6). Compared to synthetic materials, hydrogel-based materials derived from, or partially derived from, natural sources can mimic the specific aspects of the tissue microenvironment and can support both cell adhesion and growth (7). Hence, these hydrogel-based materials can provide a more favorable matrix for the growth and differentiation of cardiomyocytes (7, 8). However, limitations of structural design and manufacturing techniques, as well as the low mechanical strength and weak processability of hydrogel-based patches, still make their clinical application challenging (3, 7, 8).

Because of the limited expansion and regeneration capacity of primary cardiomyocytes, the use of human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs) provides a continuous cell source by which to produce terminally differentiated cells and avoid controversial ethical issues in biomedical research (9, 10). Although several studies have been performed with hiPSC-CMs to generate functional cardiac tissue constructs (11, 12), more studies are required to explore the interaction between hiPSC-CMs and the matrix microenvironment (i.e., scaffolds and other cells) for therapeutic improvement. Hence, further studies should focus on exploring material bioactivity, architectural design and manufacturing, the biomechanical properties of tissue constructs, and the long-term in vivo development of these tissue constructs, which will ultimately affect three-dimensional (3D) cell assembly and neotissue remodeling for clinical research purposes (2, 10, 13).

In this study, a 4D hydrogel-based cardiac patch was developed with a specific smart design for physiological adaptability (or tunability) using a beam-scanning stereolithography (SL) printing technique. Beam-scanning SL printing offers an effective methodology for creating microfabricated tissue constructs with photocurable hydrogels, which are able to achieve many essential requirements in manufacturing tissue micropatterns and macroarchitectures (14). The printing speed and laser intensity are able to be varied as required, which provides the ability to tailor the cross-linking degree of the inks and, therefore, affects the physicochemical properties of hydrogels. Moreover, it was observed that a light-induced graded internal stress, followed by a solvent-induced relaxation of material, drove an autonomous 4D morphing of the objects after printing (15, 16). It was found that this self-morphing process was able to achieve conformations that were nearly identical to the surface curvature of the heart. Moreover, taking the physiological features of the cardiac tissue and the physical properties of the hydrogel into account, a highly stretchable microstructure was created to allow for an easy switch of fiber arrangement from a wavy pattern to a mesh pattern, in accordance with the diastole and systole in the cardiac cycle. The specific design was expected to increase the mechanical tolerance of the printed hydrogel and to decrease the unfavorable effect of hiPSC-CM residence on the printed patches when exposed to the dynamic mechanics. By triculturing cardiomyocytes, mesenchymal stromal cells, and endothelial cells, the printed microfibers with specific nonlinear microstructures could reproduce the anisotropy of elastic epicardial fibers and vascular networks, which plays a crucial role in supporting the effective exchange of nutrients and metabolites, as well as guiding contracting cells for engineered cardiac tissue.

The cardiac muscle fibers mainly consist of longitudinally bundled myofibrils (cardiomyocytes and collagen sheaths), which are surrounded by high-density capillaries (17, 18). This anisotropic (directionally dependent) muscular architecture results in the coordinated electromechanical activity of the ventricles, which involves the directionally dependent myocardial contraction and the propagation of the excitation wave (19, 20). As can be observed by diffusion tensor imaging (DTI) (21, 22), a helical network of myofibers in the LV is organized to form a sheet structure, and the orientation of the fiber angles varies from approximately +60 to 60 across the ventricular wall (Fig. 1A). The visualization of the fiber structure illustrates the left-handed to the right-handed rotation of the fibers going from the epicardium to the endocardium in the LV (21). Computer-aided design (CAD)driven 3D printing offers a promising technique by which to transform the anatomical detail of cardiac fiber maps into a highly complex arrangement of fibers within an engineered cardiac tissue (23). Figure 1B shows that the spiral arrangement of 3D myocardial fibers crosses the ventricular wall (a left-handed to right-handed spiral of the fibers going from the epicardium to the endocardium) and their 2D mesh pattern projections at different angles.

(A) Photograph of the anatomical heart and the fiber structure of the LV visualized by DTI data. (B) Schematic illustration of a short-sectioned LV that illustrates the variation of fiber angle from the epicardium to the endocardium. The orientation (2D mesh pattern projection) of the fiber angles varies continuously with the position across the wall and distribution changes from the apical region to the basal region. (C) Curvature change of cardiac tissue at two different phases (diastole and systole) of the cardiac cycle, which occurs as the heartbeat and pumping blood. (D) CAD design of 3D stretchable architecture on the heart. It provides dynamic stretchability without material deformation or failure when the heart repeatedly contracts and relaxes. (E) Representation of a simplified geometric model of the fibers in the printed object. In the selected region, the angle (), the length of fiber (L), spatial displacement (D) and the ventricular curvature () are defined with systole (1) and diastole (2) states. (F) Mechanism of the internal stress-induced morphing process. Uneven cross-linking density results in different volume shrinkage after stress relaxation. Photo credit: Haitao Cui, The George Washington University (GWU).

Moreover, another specific feature of the cardiac tissue is the diastole and systole in the cardiac cycle induced by the contraction of cardiac muscle, which generates the force for blood circulation (8, 24). When taking the volume change of the heart into account, the arrangement of fibers is dynamically stretched in a selected region (Fig. 1C). Hence, the mesh pattern was changed to hexagon or wavy pattern in the 2D plane to adapt the change of ventricular curvature (Fig. 1D). It was able to create a highly stretchable structure with very limited deformability, which is expected to decrease the negative effect on the attached, susceptible cardiomyocytes. To mathematically characterize this design, we simplified the solid geometric model with a plane curve prototype to elaborate on the relationship between the redundant length of the stretchable structure (L) and the ventricular curvature () in the systole state (Fig. 1E). By the calculation, it can be estimated asL=cos1(1D22/2)D(1)where L is the redundant length of the stretchable structure from straight to curve, is the ventricular curvature in the systole state, and D is the approximate length of fiber in the diastole state (here, D = 400 m from our study). In the previous study, a light-induced 4D morphing phenomenon was demonstrated when using our customized beam-scanning SL printer (15, 16). The laser-induced graded internal stress, introduced through the printing process, is a major driving force of this 4D dynamic morphing (15, 16, 25). The uneven cross-linking density of photocrosslinkable inks generates the difference of modulus between the upper and lower surfaces of thin objects due to laser energy attenuation, leading to different volume shrinkage after stress relaxation (15). However, when multilayers were printed, the beam-scanning SL printing resulted in the repeated cross-linking of previous layers. The bottom layers had a higher cross-linking density. In this case, the bottom layer, which was cured the earliest, adhered to the substrate and could not shrink freely, while the top layer during printing could gradually and spontaneously shrink because of the release of internal stress. Thus, the printed objects have a tendency to bend toward the newly cured layer. We also found the humidity-responsive, reversible 4D phenomenon, which is swelling-induced stretching and dehydration-induced bending (15). After printing, the printed patch can transform from a 3D flat pattern to the 4D curved architecture when appropriate printing parameters are selected (Fig. 1F), which will be elaborated upon in the next section. It was hypothesized that by integrating a unique 4D self-morphing ability within the construct, the structural expandability of the design would improve the physiological adaptability of the engineered cardiac patch to the heart for in vivo cardiac regeneration.

A gelatin-based printable ink consisting of gelatin methacrylate (GelMA) and polyethylene glycol diacrylate (PEGDA) was used to create the anisotropic cardiac patch with myocardial fiber orientation. As a chemical derivative of gelatin [gelatin is derived from the hydrolysis of collagen, which is a major component of the extracellular matrix (ECM)], GelMA is a photocurable biomaterial with many arginine-glycine-aspartic acids and other peptide sequences that can significantly promote cell attachment and proliferation (14). The PEGDA solution was mixed with GelMA to decrease the swelling volume and to increase the mechanical modulus and structural stability of the printed hydrogels. The structural characteristics and mechanical properties of the printed hydrogels were determined by fiber design, printing parameters, the ink concentration, and mixing ratio of GelMA and PEGDA. To optimize the fiber design, stacked wavy architectures were generated with fiber width of 100, 200, and 400 m, fill density of 20, 40, and 60%, and fiber angles () of 30, 45, and 60 between each layer with two, four, and eight layers, respectively, using 10% GelMA and 10% PEGDA. The laser intensity, working distance, ink volume, and temperature were set to the same conditions as our previous studies (15, 26, 27) to eliminate the effect of the printing parameters. In this situation, the printing speed of the laser-based SL printing affects the photocuring performance, the structural accuracy (fineness), and the curvature of the 4D self-morphing. To ensure the complete solidification of the inks, a printing speed of 10 mm/s was set on the basis of our previous trials. By varying the printing speed (cross-linking density), a series of 4D self-morphing patches (wave pattern) were obtained with different curvatures. The mesh-patterned patches also exhibited a similar 4D morphing behavior. In all 4D self-morphing structures, the degree of deformation largely depends on the swelling, water content, and ionic strength. After 4D morphing, the wave-patterned patches maintained their wavy structure with a slight deformation. In our study, the bending of macrostructure does not significantly affect the microstructure. Figure 2A shows the curvature change of 4D morphing with increasing printing speed. Similar to the 4D morphing mathematical model by stress relaxation in the previous study (15), the relationship between the 4D curvature and printing speed can be modeled with the materials and printing parameters using the following equation1/r=4.7802.53lnv[mm1](2)where r is the radius of the object curvature after 4D morphing, is the printing speed (millimeters per second), and 0 is the shrinkage, which is dependent on both the material and the immersion medium. Here, 0 = 0.012 s1 in aqueous solution. The results demonstrated that the patches printed with a print speed (6 mm/s) had an appropriate curvature with the 4D morphing to obtain a sufficient integration with the LV surface of the mouse hearts.

(A) Curvature change of 4D morphing versus printing speed (means SD, n 6, *P < 0.05). (B) Printing accuracy of the hydrogel patches versus fiber width for different fill density (fd; means SD, n 6, *P < 0.05, **P < 0.01, and ***P < 0.001). (C) Color map of tensile moduli of the patches with varying GelMA and PEGDA concentrations. (D) Optical and 3D surface plot images of the patches. Scale bars, 200 m. (E) Average elasticity values of the wave-patterned patches in horizontal (x) and vertical (y) directions. Number sign (#) shows the statistical comparison between the horizontal and vertical directions (means SD, n 6, **P < 0.01 and ##P < 0.01). (F) Uniaxial tensile stress-strain curves of 5% GelMA and 15% PEGDA. Immunostaining of cell morphology (F-actin; red), sarcomeric structure (-actinin; green), gap junction [connexin 43 (Cx43); red], and contractile protein [cardiac troponin I (cTnI); red] on the patches on (G) day 1 and (H) day 7. Scale bars, 20 m. (I) Beating rate of hiPSC-CMs on the patch and well plate on day 3 and day 7 (means SD, n 6, *P < 0.05; n.s. no significant difference). BPM, beats per minute. Photo credit: Haitao Cui, GWU.

As is shown in Fig. 2B, the printing accuracy of different fiber width, fiber angle, layer number, and fill density of the fiber arrangement was investigated. The fiber pattern with a 100-m width showed significantly lower accuracy (50%) when compared to both fibers with 200-m (>70%) and 400-m (>90%) widths. In addition, the fiber pattern with a 60% fill density showed lower accuracy than the fiber pattern with 40% fill density. This implies that the fiber pattern with higher fill density or lower width is associated with more directional changes of the laser head per unit area, which is a function of the limitation of the printing resolution. In addition, there was no significant difference in the accuracy observed when increasing the number of stacked fibers (or fiber angles) due to the high reproducibility of the SL printing (fig. S1A). It was observed that smaller fiber widths or higher fill densities had a higher surface area per unit area, which was beneficial for the attachment of more cells, as the increased surface area better mimics the native myofibers. According to a previous study, the quantitative measurement of fiber angles showed that the dominant distribution of fiber angle was +45 to 45 from the epicardium to the endocardium (21). Therefore, a fiber pattern was printed with a 200-m width, 40% fill density, and a maximum angle of 45 for adjacent layers to optimize the mechanical properties of the hydrogel patches.

To test and measure the mechanical (both compression and tensile) modulus of the hydrogels, we varied the mixed weight ratio of GelMA and PEGDA from 5 to 20% (Fig. 2C and fig. S1, B and C). The results demonstrated that the mechanical moduli of the hydrogels fall within the range of the native myocardium modulus (101 to 102 kPa) in the physiological strain regime (28, 29). In addition, swelling testing showed that when GelMA was mixed with PEGDA, the printed hydrogels maintained excellent structural stability without notable swelling (fig. S1D). With consideration for the optimized ink viscosity and hydrogel elasticity, the inks used to fabricate our myofiber patches were formulated with concentrations of 5% GelMA and PEGDA (5, 10, and 15%) and were effectively printed on the basis of our design. Figure 2D shows the optical and 3D surface plot images of the patches printed by 5% GelMA and 15% PEGDA with a 200-m width, 40% fill density, and a 45 angle for adjacent layers. The fluorescent images of the 3D printed patches are also displayed (fig. S2A). The anisotropic behaviors of the wavy-patterned patches in the horizontal (x) and the vertical (y) direction demonstrated that uniaxial tension on the fiber pattern resulted in different deformation and stress generation in a directionally dependent manner (Fig. 2E). In particular, the stress-strain curve of the patch with 5% GelMA and 15% PEGDA was consistent with the tensile features of the native myocardium within the physiological strain regime (Fig. 2F) (19, 30). The fatigue was obvious along the y direction at the initial stage, which is attributed to the lower connectivity of fibers in the y direction and higher extendibility of the wavy-patterned fibers in the x direction. It is expected that this physiologically adaptable design would increase the stretchability and stability of the hydrogel patches, allowing them to absorb and release energy against the force of cardiac contraction (31, 32). Compared to the mesh design, the current architecture would allow for structural compliance of the hydrogel fibers without notable deformation. Moreover, the successfully printed patterns also well represent the microstructure of the native myocardial tissue, which is formed from collagen fibers and other ECM proteins, together with cardiomyocytes. However, the width of the myofibers within the myocardial tissue was much smaller (30 to 40 m) than the printed pattern (200 m), which is largely a limitation of the resolution of the currently available technology.

After the optimization of both the printing parameters and the ink formulation, the cardiac patches were manufactured with 5% GelMA and 15% PEGDA using a beam-scanning SL printing system. The wavy-patterned patches with a diameter of 8 mm and a thickness of 600 m were used to perform the in vitro studies, while the mesh-patterned patches of the same fiber volume fraction served as the control. By keeping the same surface area across the different construct patterns, we could ensure that there would be the same available cell number for each of the patches. Upon analysis, the redundant length (L) of the stretchable structure was determined to be around 140 m. Because of their capacity for restoring cardiac function in previous studies, hiPSC-CMs were cultured using the same protocol developed at the National Heart, Lung, and Blood Institute (NHLBI) (33). Before cell seeding, a thin layer of Matrigel was precoated on the well plate or patches surface to improve the hiPSC-CM adhesion. By day 7, spontaneous contractions of monolayer hiPSC-CMs were observed (fig. S2B and movie S1), and immunostaining results demonstrated that hiPSC-CMs displayed specific myocardial protein expression, including sarcomeric alpha-actinin (-actinin), connexin 43 (Cx43), and cardiac troponin I (cTnI). (fig. S2, C and D). The cell-laden ink was printed by mixing 1 106 per ml of hiPSC-CMs with 5% GelMA and 15% PEGDA. However, a decrease in the metabolic activity of the hiPSC-CMs was observed, and the distinct cardiac beating behavior was not evident (fig. S2, E and F). These observations were likely the result of the limited 3D space within the hydrogel. Hence, a postseeding approach was then applied to fabricate the cardiac patches. Compared to the cell-laden samples, the hiPSC-CMs seeded on the patches showed significantly higher proliferation and beating rate. The attached hiPSC-CMs exhibited spontaneous contractions along the fibers on day 3 (movie S2). Moreover, the immunostaining images revealed robust F-actin, -actinin, Cx43, and cTnI expression of hiPSC-CMs on the printed patches (Fig. 2G). After 7 days of culture, the hiPSC-CMs began to form aggregation structures atop the printed fibers and began to contract synchronously across the entire patches, indicating electrophysiological coupling of the cells (Fig. 2H). Moreover, the beating rate of the hiPSC-CMs on the printed patch was notably similar to that of the monolayer hiPSC-CMs on the seeded well plate (Fig. 2I).

According to previous studies, human mesenchymal stromal cells (hMSCs) have been widely used in coculture with cardiomyocytes and endothelial cells to improve cell viability, myogenesis, angiogenesis, cardiac contractility, and other functions due to their paracrine activity (34, 35). Hence, a triculture of hiPSC-CMs, human endothelial cells (hECs), and hMSCs was performed to fabricate the vascularized cardiac patches. The analysis of cell tracker staining was conducted to investigate the distribution of different cells in the triculture and to optimize the cell ratio in the triculture system based on the calculated fluorescent value. The results demonstrated that when the initial ratio of seeded cells was 4:2:1, the resultant cellular proportion of hiPSC-CMs, hECs, and hMSCs was ~ 30, ~40, and ~30%, respectively, at confluency, which falls within the range of the cellular composition [25 to 35% cardiomyocytes, 40 to 45% endothelial cells, and ~30% supporting cells (i.e., fibroblasts, smooth muscle cells, hematopoietic-derived cells, and others)] of the human heart (Fig. 3A) (36, 37). After 7 days of culture, the printed construct showed a uniform cell distribution and longitudinal alignment of the cells along the fiber direction (Fig. 3B and fig. S3). Autofluorescence images of green fluorescent proteintransfected (GFP+) hiPSC-CMs on day 7 indicated that the cardiomyocytes exhibited an increased proliferation rate on the patches, as compared to initial seeding on day 1, and were able to generate spontaneous contractions (Fig. 3C and fig. S4). After 7 days of culture, fluorescent image analysis of CD31 [platelet endothelial cell adhesion molecule-1 (PECAM-1)] stained patches revealed that the wave-patterned patch had a higher density of capillary-like hEC distribution along the fibers when compared to the mesh control (Fig. 3D). In our previous studies, we found that the beam-scanning laser is able to cure the ink for the macroarchitectural formation together with the aligned microstructure present on the printed fibers (16). Hence, the hECs were easily grown along the fiber direction. Moreover, the iPSC-CMs exhibited an excellent contraction-relaxation behavior along the fibers in the wave-patterned patches, potentially allowing for a local mechanical stimulation on the fiber resident cells, which can help to improve the growth and distribution of hECs. In addition, immunostaining analysis of the cTnI and the marker von Willebrand factor (vWf) indicated that our wave-patterned patches contained a dense network of vascular cells interwoven with hiPSC-CMs distributed over the printed fibers, and the ratio of hiPSC-CMs and hECs was largely retained with 45% hiPSC-CMs (Fig. 3, E and F). Furthermore, it has been well established that the electrical activity at the cardiomyocyte membrane is controlled by ion channels and G proteincoupled receptors, which are usually actuated by calcium transients (38). The electrophysiological profiles of the cardiac patches demonstrated the generation of typical calcium oscillation waveforms and synchronous beating along with the printed fibers across the entire patches after 3 days (Fig. 3G). Over the next 7 days of culture, the amplitudes of calcium transients gradually increased to a stable state, suggesting the establishment of excellent functional contraction-relaxation and electrophysiological behaviors (Fig. 3, H and I).

Cell distribution of tricultured hiPSC-CMs (green), hECs (red), and hMSCs (blue) on the cardiac patches using cell tracker staining after (A) 1 day of confluence and (B) 7 days of culture. Scale bars, 200 m. (C) Autofluorescence 3D images of GFP+ hiPSC-CMs on the wave-patterned patch on day 1 and day 7. Scale bars, 100 m. (D) Immunostaining of capillary-like hEC distribution (CD31; red) on the hydrogel patches. Scale bars, 200 m. Immunostaining (3D images) of cTnI (red) and vascular protein (vWf; green) on the (E) wave-patterned and (F) mesh-patterned patches. Scale bars, 200 m (3D image) and 20 m (2D inset). Calcium transients of hiPSC-CMs on the hydrogel patches recorded on (G) day 3 and (H) day 7. (I) Peak amplitude of the calcium transients of hiPSC-CMs on the mesh- and wave-patterned patches on day 3, day 7, and day 10 (means SD, n 30 cells, *P < 0.05).

To enhance the effectiveness of our design, a custom-made bioreactor consisting of a dynamic flow device and a mechanical loading device was constructed to provide a physiologically relevant environment, which could incorporate both mechanical strain and hydrodynamics (Fig. 4A) (39). The patches were compressed in the radial direction using positive pressure between the piston and stationary polydimethylsiloxane (PDMS) holder to yield a mechanical loading, which mimics the contractile behavior of the in vivo human heart (fig. S5A). During the dual mechanical stimulation (MS), the applied force was stored in the patch as strain energy, which was then responsible for returning the patch to its original shape. The out-of-plane loading (bending) determines the stretch and recovery of the fibers, while the fluid shear stress regulates cellular orientation (Fig. 4B). Both were applied to the patches and transferred onto the cells to improve the vascularization and myocardial maturation of the resident cells.

(A) Schematic illustration of a custom-made bioreactor to apply dual MS for the maturation of engineered cardiac tissue. PMMA, polymethylmethacrylate. (B) Both the out-of-plane loading and fluid shear stress applied to the patches. (C) Immunostaining of cTnI (red) and vWf (green) on the wave-patterned patch under MS condition (+MS) versus nonstimulated control (MS). Scale bars, 50 m. (D) Immunostaining of the -actinin (green) and Cx43 (red) on the wave-patterned patch under MS condition (+MS) versus nonstimulated control (MS). Scale bars, 20 m. (E) Cross-sectional immunostaining of the sarcomeric structure (Desmin; green) and vascular CD31 (red) on the patches under MS condition (+MS). Scale bars, 50 m. (F) The beating rate of hiPSC-CMs on the printed patches under MS condition (+MS) versus nonstimulated control (MS) on day 14 (means SD, n 6, *P < 0.05). BPM, beats per minute. Relative gene expression of (G) myocardial structure [myosin light chain 2 (MYL2)], (H) excitation-contraction coupling [ryanodine receptor 2 (RYR2)], and (I) angiogenesis (CD31) on the patches under MS condition (+MS) versus nonstimulated control (MS) on day 1, day 7, and day 14 (means SD, n 9, *P < 0.05, **P < 0.01, and ***P < 0.001).

After 2 weeks of dynamic culture, we observed a higher expression of mature cardiomyogenic cTnI and angiogenic vWf in MS samples and more longitudinally aligned vascular cells, when compared to the nonstimulated control (Fig. 4C and fig. S5, B and C). In addition, the patches exhibited enhanced sarcomere density and junctions, as identified by the -actinin and Cx43 expression of the hiPSC-CMs (Fig. 4D and fig. S5, D and E). Cross-sectional images illustrated that the high density of cell assemblies on the wave-patterned patches was evident under MS conditions and these assemblies exhibited a higher expression of desmin and CD31 markers compared to the mesh control (Fig. 4E). This suggests that the specific design of the cardiac patch was able to impede the mechanical force against material deformation in our dynamic system to support repeatable stretch cycles and decrease the negative effect on the cells. Moreover, the assembled hiPSC-CM fibers on the cardiac patch spontaneously and synchronously contracted along with the fiber direction (Fig. 4F and movie S3). However, the entire patch did not exhibit in-plane contraction or macroscopic movement itself due to the high mechanical resistance of the hydrogel material. In general, it was observed that the wave-patterned patch was capable of stretching to a physiologically relevant fiber pattern compared to the mesh design, which could improve cell guidance and elongation along the fiber direction.

Consistent with the immunostaining results, the expression of cardiac-related genes, including genes associated with sarcomeric structure, excitation-contraction coupling, and angiogenesis, was significantly increased on day 14 compared to day 7. These results suggest that there was an increase in maturation of the iPSC-CMs on the printed patches over time (Fig. 4, G to I, table S1, and fig. S6). After the application of the MS, the expression of the MYL2 (myosin light chain 2) and RYR2 (ryanodine receptor 2) genes were significantly increased in our wave-patterned patches on day 14, as compared to the mesh control. This demonstrates that our specific patch design can enhance iPSC-CM contractile and electrical function under MS. Moreover, the angiogenic CD31 gene was also considerably up-regulated on the wave-patterned patches with perfusion culture. In general, the gene expression on day 14 was up to 28-fold higher compared to day 1, and an average of 5.5-fold increase in the expression of maturation genes was observed with the MS condition as compared to the nonstimulated groups. This observation provides further evidence that significantly enhanced cardiac maturation is achievable on the printed patches when specific structural design and physiologically relevant culture conditions are combined.

Having used the dynamic culture system to enhance the maturation of hiPSC-CMs in vitro, we further investigated the vascularization and myogenic maturation of the printed cardiac patches in vivo. Ischemia-reperfusion (I/R) is a major contributor to the myocardial damage resulting from MI in humans (40). Murine models of I/R injury provide an effective means to simulate clinical acute or chronic heart disease for cardiovascular research (41, 42). Hence, a chronic MI model with I/R injury was created to assess the functional effects of cardiac patch implantation (43, 44). The cellularized and acellular patches were implanted onto the epicardium of immunodeficient nonobese diabetic severe combined immunodeficient gamma (NSG) mice and were assessed for long-term development 4 months after implantation. Compared to the classic MI model, our I/R injury model produced a shortened recovery time, less inflammation, and higher survival rates. The patches (4-mm diameter by 600-m thickness in size) were entirely positioned over the infarcted (ischemia) site of the mouse hearts (Fig. 5, A and B, and movie S4). To assess the direct interaction (structure and cells) between the patch and the host epicardium, we did not apply fibrin glue. After 3 weeks of implantation, optical images showed that the cellularized patches had a firm adhesion to the epicardium regardless of the contractile function of the heart (Fig. 5C). Hematoxylin and eosin (H&E) assessment confirmed the robust epicardial engraftment of the cell-laden patches, which contained high-density cell clusters after 3 weeks (Fig. 5D). Fluorescent images also showed that the GFP+ hiPSC-CMs (green) maintained higher viability after 3 weeks of implantation (Fig. 5E). The immunofluorescence analysis of cTnI and vWf illustrated the existence and development of hiPSC-CMs and hECs on the cellularized patches in the treated region with time. The image results showed that many vascular cells were found spanning the interface of the patch and myocardium and expanded within the myocardial patch (Fig. 5F).

(A) Optical image of surgical implantation of the patch. (B) Optical image of a heart I/R MI model after 4 months. (C) Optical image of the implanted cellularized patch at week 3, exhibiting a firm adhesion (inset). (D) H&E image of the cellularized patch at week 3, demonstrating the cell clusters with a high density (yellow arrowhead). Scale bar, 400 m. (E) Fluorescent image of (GFP+) iPSC-CMs on the patch at week 3, showing a high engraftment rate (yellow arrowhead). Scale bar, 100 m. (F) Immunostaining of cTnI (red) and vWf (green) on the cellularized patch at week 3. Scale bar, 100 m. (G) H&E images of mouse MI hearts without treatment (MI) and with cellularized patch (MI + patch) at week 10. Infarct area after MI (yellow circles). Scale bars, 800 m. (H) Cardiac magnetic resonance imaging (cMRI) images of a mouse heart with patch at week 10. Left (spin echo): the position of the heart and implanted patch. Right (cine): the blood (white color) perfusion from the heart to the patch. Photo credit: Haitao Cui, GWU.

After 10 weeks of implantation, H&E staining results showed that the infarct sizes of the patch groups (~3.8 0.7%) were smaller than the MI-only control (~8.4 1.1%), suggesting that the patch can provide mechanical support to effectively prevent LV remodeling (Fig. 5G and fig. S7A). The images and videos of the cardiac magnetic resonance imaging (cMRI) demonstrated that the implanted patch was able to contract and relax with the heartbeat of the mouse and also confirmed its excellent structural durability along with evident blood perfusion from the heart to the patch (Fig. 5H and movie S5). Fluorescent images also showed that the GFP+ hiPSC-CMs (green) maintained higher viability after 10 weeks of implantation (fig. S7B). hiPSC-CMs with cTnI+-expressing capillaries (vWf+) were observed in the patches, where the lumen structure of neovessels was also clearly visible (Fig. 6A). Together, these results indicated that epicardially implanted patches exhibited robust survival and vascularization in vivo. Moreover, a high density of capillaries identified by human-specific CD31 expression was observed within the cellularized patches, suggesting that the implanted hECs and hMSCs increased the vessel formation throughout the patch in vivo (Fig. 6B).

(A) Immunostaining of cTnI (red) and vWf (green) on the cellularized patch at week 10. Scale bar, 800 m. Border of the heart (white dashed line) and capillary lumen (white arrow). Scale bar (enlarged), 100 m. (B) Immunostaining of human-specific CD31 (red) on the cellularized patch at week 10, showing the generated capillaries by hECs. Scale bar, 800 m. Border of the heart (white dashed line). Scale bar (enlarged), 100 m. (C) Immunostaining of cTnI (red) and vWf (green) on the cellularized patch for 4 months, showing the increased density of the vessels. Scale bar, 800 m. Border of the heart (white dashed line) and capillary lumen (white arrow). Scale bar (enlarged), 100 m. (D) Quantification of capillaries with vWf staining data for 10 weeks and 4 months (means SD, n 6, *P < 0.05 and **P < 0.01). (E) Immunostaining of human-specific CD31 (red) on the cellularized patch for 4 months. Scale bar, 800 m. Border of the heart (white dashed line). Scale bar (enlarged), 100 m. (F) Immunostaining of -actinin (green) and human-specific CD31 (red) on the cellularized patch at month 4. Scale bars, 50 m.

By 4 months, H&E staining results showed a higher cell density and smaller infarct area (~5.6 1.5%) in the cellularized patch compared to the acellular patch and MI groups (~14.3 2.3%; fig. S7C). The GFP+ fluorescent results demonstrated that the hiPSC-CMs retained high engraftment rates (fig. S7D). Similar to what was observed at 10 weeks, the cellularized patch had a strong integration within the epicardium, whereas the cell-free patch had a weak adhesion by month 4. In addition, the cMRI images also illustrated that the implanted patch had an excellent connection with the mouse heart (fig. S7E). The positive expression of mature cTnI further indicated the presence of advanced structural maturation of the hiPSC-CMs in the treated region, and the capillaries were also substantially identified by the vWf staining in the cell-laden patch groups (Fig. 6C). The images also demonstrated more progressive implant vascularization with a 1.5-fold increase in blood vessel density after 4 months of implantation, when compared to cell-free controls (Fig. 6D). The data were counted by five randomly selected fields in each heart. However, the fraction of humanized vessels was not significantly increased, as identified by the human-specific CD31 staining (Fig. 6E). Therefore, the increased vascularization and vascular remodeling in vivo likely originated from the host vessel ingrowth as opposed to the implanted human vessels at the initial stage of implantation. However, differing from the in vitro results, the cross-sectional images of the implants did not exhibit substantial sarcomeric structure (identified by -actinin) when compared to the native cardiac tissue (Fig. 6F). It can be observed that the cell aggregation in the vertical direction showed a disordered assembly and 3D stacking behavior. Overall, these results demonstrated that the printed patches underwent progressive vascularization, largely remained on the epicardial surface of the LV over the 4-month implantation period, and effectively covered all of the infarcted area. Cardiac function was also evaluated at different time points after injury via cMRI assessments. The LV ejection fraction of all patch groups (~64.1 3.5%) was higher than the MI-only group (~56.1 1.5%); however, there was no difference observed between the cellularized patch group and the cellular group.

MI is a leading cause of morbidity and mortality worldwide. The hiPSC-CMs provide a potentially unlimited source for cardiac tissue regeneration, as they are able to recapitulate many of the physiological, structural, and genetic properties of human primary cardiomyocytes and heart tissue (13). Current methods for the treatment of MI largely involve injecting cardiomyocytes directly into the epicardial infarct zone; however, because of the limited engraftment capacity of the injected cardiomyocytes, injection therapies are not fully satisfactory in restoring cardiac functionality (13). Several studies have been performed with hiPSC-CMs to generate functional cardiac tissue constructs using tissue engineering techniques (7, 10). However, the physiological features of hiPSC-CMs are more sensitive to the physicochemical and bioactive properties of the scaffolds in which they reside. Compared to synthetic polymers, natural polymer-based hydrogels can provide a more favorable matrix for the growth and differentiation of cardiomyocytes (7). However, limitations of structural design and manufacturing techniques, as well as the low mechanical strength and weak processability of hydrogel-based patches, still make their clinical application challenging.

As a proof of concept, a physiologically adaptable 4D cardiac patch, which recapitulates the architectural and biological features of the native myocardial tissue, has been printed using a beam-scanning SL printing technique. The smart patches provided mechanical support, a physiologically tunable structure, and a suitable matrix environment (elasticity and bioactivity) for cell implantation. Successful vascularization of the patches allowed for the continued metabolic demand of hiPSC-CMs and permitted them to remain both viable and functional throughout the in vivo study. Robust engraftment and development of the implanted patches were further confirmed using a more clinically relevant and mechanically realistic environment. The study results showed that the anisotropic mechanical adaption of the printed patches improved the maturation of cardiomyocytes and vascularization in vitro under MS. After implantation into the murine MI model, the printed patches exhibited high levels of in vivo cell engraftment and vascularization.

In a previous study, an engineered auxetic design was developed to give a cardiac patch a negative Poissons ratio, providing it with the ability to conform to the demanding mechanics of the heart (31). Here, we further propose and develop a 4D physiologically adaptable design for a cardiac patch, which includes hierarchical macro- and microstructural transformations attuned to the mechanically dynamic process of the beating heart. Therein, a physiological adaptation is evident in the response of cells (or genes) to the microenvironmental change. Similarly, the adaptive responses of the resident cells on the scaffolds to replicate the native microenvironment are crucial for the in vivo integration of engineered tissue with and the host tissue after implantation. In addition, a highly biomimetic in vitro culture system was developed with dynamic perfusion and mechanical loading to enhance cardiac maturation. Overall, the current work has several unique features: (i) a greatly improved mechanical stretchability of the hydrogel patches; (ii) a triculture of hiPSC-CMs, hMSCs, and hECs, which is necessary to obtain a complex cardiac tissue; (iii) an application of in vitro dual MS by which to improve cardiac maturation; and (iv) in vivo long-term development of the printed patches in a murine chronic MI model to evaluate the potential therapeutic effect.

Although several studies have shown that cell transplantation can greatly improve cardiac function in the MI model, no substantial evidence supporting these improvements in cardiac function was found in the current study. In the MS culture studies, it was observed that the entire patch did not exhibit in-plane contraction. The high mechanical resistance of the hydrogel could be a reason as to why the patches did not significantly enhance cardiac function in vivo. Moreover, as is known, the implanted human cardiomyocytes exhibit different beating frequencies and other biological features within the host mouse heart (13, 45, 46). Hence, integrated functional repair was not observed in this study. Differing from the hypothesis of the functional enhancement, the in vivo results revealed that the enhanced cardiomyogenesis and neovascularization of humanized patches did not significantly improve the cardiac function of the MI mice. The patches provided cellularized niche conditions so that most of the implanted cardiomyocytes were alive, although they still exhibited immature 3D sarcomeric organization. Moreover, the neovascularization effects of the cell-laden patches at the infarct region were also confirmed, suggesting that paracrine effects appear to be a major contributing factor. Although there was a lack of functional integration between humanized patches with the hearts of the host mice, the printed patches exerted no adverse effects on the host cardiac function or vulnerability to arrhythmias. The cell transplantation improved the cellularized environment in the infarct area by, at least in part, promoting angiogenesis and increasing cell retention. The multiple cell transplantation with a high density of hiPSC-CMs retention in the murine MI model suggests that this goal may have been at least partially achieved. While applying humanized grafts to infarcted rodent hearts would likely confirm the paracrine effects, large animal studies are warranted to further evaluate the therapeutic efficacy toward a potential clinical use.

In the future, a physiologically relevant large animal study, such as a porcine or nonhuman primate MI model, will serve as a more realistic means to study cardiac patch engraftment (42, 46). Moreover, developments in advanced printing techniques will enable the development of thick, scale-up ready myocardial tissue, which will play a prominent role in the ultimate success of clinical cardiac engineering therapies. In general, the developed cardiac patch has a great potential to provide a desired therapeutic effect on the in vitro maturation and in vivo retention of hiPSC-CMs, based on the previously unidentified engineering design and manufacturing process.

These studies were designed to evaluate the concept of a 4D cell-laden cardiac patch with physiological adaptability as a potential method for the treatment of MI. To evaluate this technology, we tricultured hiPSC-CMs, hMSCs, and hECs to obtain a complex cardiac tissue and also applied in vitro dual MS to replicate the physiologically relevant conditions for the improvement of regenerated myocardial function. A 4-month in vivo study was conducted to assess the performance of our 4D cell-laden cardiac patches, where the animals were randomly assigned to different experimental groups before the experiments. The sample size and power calculation were determined on the basis of our experience with the experimental models and the anticipated biological variables. Typically, the power is 0.8, and the significance level is 0.05 when the effect size is determined by the minimum sample difference divided by the SD (GPower 3.1). All experiments were blinded and replicated. The sample sizes and replicates are shown in the figure legends.

Ten grams of gelatin (type A, Sigma-Aldrich) was dissolved in 100 ml of deionized water with stirring at 80C. Next, 5 ml of methacrylic anhydride was added dropwise into the gelatin solution. After reaction at 80C for 3 hours, the reactant was dialyzed in deionized water for 5 days at 40C to remove any excess methacrylic acid. The GelMA solid product was finally obtained through lyophilization. The ink solutions consisted of GelMA [with concentrations of 0, 5, 10, 15, or 20 weight % (wt %)], 1 wt % 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, photoinitiator), and PEGDA (Mn = 700; Sigma-Aldrich; with concentration of 0, 5, 10, 15, and 20 wt %). Solutions were prepared using a 1 phosphate-buffered saline (PBS) solution.

Our 3D computational myocardium model was programmed on the basis of DTI results and was simplified to the basic geometry to replicate the fiber orientation of the native myocardium. The wave (or hexagonal) microstructure was configured with different diameter fibers and fiber angels between each layer, while the mesh microstructure served as a control. To optimize the fiber design, wave or mesh architectures with side lengths of 100, 200, and 400 m and internal angles () of 30, 45, and 60 between each layer were designed with two, four, and eight layers, respectively. All cardiac construct models were saved as .stl files, processed using the Slic3er software package, and were transferred to the 3D printer. Representative CAD models of the constructs were calculated and predicted for surface area, porosity, and other structural characteristics.

Printed cardiac patches were manufactured using our customized table-top beam-scanning SL printer, which is based on the existing Printrbot rapid prototyping platform. This system consists of a movable stage and a 110-m fiber optic-coupled solid-state ultraviolet (355 nm) laser mounted on an X-Y tool head for three-axis motion. The laser scans and solidifies the top layer of ink in a reservoir, and a movable platform lowers the construct further into the ink, covering it with the next material layer. For this study, the effective spot size of the emitted light was 150 50 m and had an energy output of ~20 uJ at 20 kHz. The ability to alter the frequency of the pulsed signal facilitates power control at the material surface ranging from 40 to 110 mW.

Different stacked architectures with fiber widths of 100, 200, and 400 m; fill densities of 20, 40, and 60%; and fiber angels of 30, 45, and 60 between each layer were manufactured with two, four, and eight layers, respectively. The printing accuracy of the patterns was quantified by the mean trajectory error (Et) compared to the designed shape. Et=1ni=0n(x(i)xt(i))2+(y(i)yt(i))2, where n 20 is the number of data points collected. Compressive and tensile mechanical properties were measured with an MTS criterion universal testing system equipped with a 100-N load cell (MTS Systems Corporation). For compressive testing, the printed patches (2 cm by 2 cm) having different microstructures were placed on the tester. The crosshead speed was set to 2 mm/min, and Youngs modulus was calculated from the linear region of the compressive stress-strain curves. For the tensile testing, the samples were mounted on to custom-made copper hooks affixed to the tester and were pulled at a rate of 1 mm/min to a maximum strain of 20%. Youngs modulus was calculated from the linear portion of the tensile stress-strain curve. In addition, the representative uniaxial tensile stress-strain plots for the latitudinal and longitudinal specimens of myocardial constructs were used to evaluate the anisotropic mechanical properties. The swelling behavior was evaluated by quantifying the weight gain after equilibrium swelling. The printed samples were immersed in PBS at 37C for 7 days. The swelling ratios of hydrogel matrices were calculated as equilibrium mass swelling ratio (SR). SR = (wt w0)/w0 100%, where w0 is the original weight of printed samples and wt is the equilibrium weight of samples after swelling.

hiPSC-CMs and GFP+ hiPSC-CMs were cultured in cardiomyocyte basic medium using the same protocol developed by our collaborators at the NHLBI (33). hECs (human umbilical vein endothelial cells; Thermo Fisher Scientific) were cultured in endothelial growth medium consisting of Medium 200 and low-serum growth supplement. hMSCs (harvested from normal human bone marrow, Texas A&M Health Science Center, Institute for Regenerative Medicine) were cultured in mesenchymal stem cell growth medium consisting of minimum essential medium, 20% fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin. All experiments were performed under standard cell culture conditions (in a humidified, 37C, 95% air/5% CO2 environment) with hECs and hMSCs of six cell passages or less.

After the patches were printed, iPSC-CMs, hECs, and hMSCs with different ratios were seeded on the patch constructs (the surface of the patch was precoated with a thin layer of Matrigel, Corning). The tricultured cardiac patches were maintained in the mixed medium at a 1:1:1 ratio for further characterization and in vitro cell study. hECs and hMSCs were prestained with CellTracker Orange CMRA Dye and CellTracker Blue CMAC Dye (Molecular Probes) and were then seeded onto the constructs. After 1, 3, and 7 days of coculture, cells were imaged using a Zeiss 710 confocal microscope. Cell proliferation on days 1, 3, and 7 were quantified using a cholecystokinin-8 solution [10% (v/v) in medium; Dojindo]. After 2 hours of incubation, the absorbance values were measured at 570 and 600 nm on a photometric plate reader (Thermo Fisher Scientific). The spreading morphology and arrangement of hMSCs and hECs were characterized using the double staining of F-actin (red, Texas Red; 1:200) and nuclei [blue, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), Thermo Fisher Scientific; 1:1000].

A customized bioreactor system consisting of a mechanical loading device and a dynamic flow device was used to culture our cell-laden constructs. The dynamic flow device is composed of four parts: a perfusion chamber, a flow controller, a nutrient controller, and a gas controller (5% CO2/95% air). The culture medium was perfused through the constructs using a digital peristaltic pump (Masterflex, Cole-Parmer) over the whole experimental period, which facilitated the efficient transfer of nutrients and oxygen. A shear stress was set at 10 dynes/cm2 (which is within the range of the shear stress observed in microcirculation), and a flow rate of 8.4 ml/min was selected (the viscosity of medium is ~7.8 104 Ns/m2) (47, 48). A PDMS holder was used to both firmly mount the patches within a polymethylmethacrylate chamber and to maintain the patch structure during cell culturing and MS to prevent undesired movement and damage. The patches were compressed at the speed of 60 times/min in the radial direction using positive pressure between the piston and the stationary holder to yield the mechanical force. The calculated (preload) contractile force per unit area was ~50 mN/mm2 along the fiber direction to match those in the native cardiac tissue. The cardiac constructs were placed in the bioreactor system and incubated at 37C for 7 and 14 days.

After 1 and 2 weeks of culture, cellular functions including cardiomyogenesis and angiogenesis were assessed using an immunofluorescence method. After the predetermined period, the cell-laden constructs were fixed with formalin for 20 min. The samples were permeabilized in 0.1% Triton X-100 for 15 min and were then incubated with a blocking solution [containing 1% bovine serum albumin, 0.1% Tween 20, and 0.3 M glycine in PBS] for 2 hours. The cells were then incubated with primary antibodies at 4C overnight. After incubation with primary antibodies, secondary antibodies were introduced to the samples in the dark for 2 hours at room temperature, followed by incubation with a DAPI (1:1000) solution for 5 min. All images were obtained using the confocal microscope, and protein quantifications were performed using ImageJ (49). In addition, the immunostaining analysis was performed with sliced fragments that were cut with a cryostat microtome. The primary antibodies that were used for our study were purchased from Abcam and included anti-actinin (1:500), antihuman-specific CD31 (human-specific PECAM-1; 1:500), anti-desmin (1:1000), anti-Cx43 (1:1000), anti-cTnI (1:500), and anti-vWf (1:1000). The secondary antibodies were purchased from Thermo Fisher Scientific and included anti-mouse Alexa Fluor 594 (1:1000) and goat anti-rabbit Alexa Fluor 488 (1:1000).

To evaluate the functional beating behavior, iPSC-CMs were observed and recorded using the inverted microscope and confocal microscopy. The Ca2+ that triggers contraction comes through the sarcolemma and plays an important role in excitation-contraction coupling of the heart beating. After the predetermined period, intracellular calcium transients were recorded under the fluorescent microscope at a wavelength of 494 nm over 30 to 120 s. Movies were analyzed with an ImageJ software to measure the fluorescence intensities for two to eight regions of interest (F) and for three to eight background regions (F0) per acquisition.

The cardiac tissue constructrelated gene expression was analyzed by a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Specifically, myocardial structure [cTnI (TNNI3), cTnT (TNNI2), MYL2, MYL7, myosin heavy chain 6 (MYH6), MYH7, and -actinin 2 (ACTN2)], excitation-contraction coupling (calsequestrin 2, RYR2, phospholamban, sodium/calcium exchanger 1, and adenosine triphosphatase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2), and angiogenic genes (vWf and CD31) were studied to detect the cardiomyocyte and vascular maturation processes in the constructs. The primers that were used are shown in the Supplementary Materials (table S1). Briefly, the total RNA content was extracted using TRIzol reagent (Life Technologies). The total RNA purity and concentration were determined using a microplate reader [optical density at 260/280 nm within 1.8 to 2.0). The RNA samples were then reverse-transcribed to complementary DNA using the Prime Script RT Reagent Kit (Takara). RT-PCR was then performed on the CFX384 Real-Time System (Bio-Rad) using SYBR Premix Ex Taq according to the manufacturers protocol. The gene expression levels of the target genes were normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The relative gene expression was normalized against the control group to obtain the relative gene expression fold values, which were calculated via the 2Ct method.

The in vivo development of the printed cellularized constructs was evaluated using a xenograft model of transplantation into 6-week-old NSG mice. All the animal experiments were approved by the Institutional Animal Care and Use Committee of the NHLBI. A method of random and blinded group allocation was applied to our animal experiments. The murine model with chronic MI was created via an I/R procedure to analyze implanted cell development, remodeling, and infarction treatment for 4 months. The printed cellularized patches (4-mm diameter by 600-m thickness in size) were prepared in sterile conditions and were surgically implanted into the LV ischemic area of each NSG mouse through a limited left lateral thoracotomy. The acellular patch and MI-only groups served as controls. At different time points after implantation, cMRI was performed to visualize the beating heart and to evaluate the structural/functional parameters, which included the ejection fraction, end-systolic volume, end-diastolic volume, stroke volume, and cardiac output, among others. Last, animals were euthanized, and the specimens, along with the adjacent tissues, were collected for further examination.

Histology was used to qualitatively examine the samples at different time points and included the examination of cellular cytoplasm, red blood cells, and cell distributions. The samples were fixed in formalin, processed, and were embedded in optimal cutting temperature compound for cryosection histology. The samples were cut into 5- to 10-m slides. The mean infarct size was also calculated through the histologic studies. The infarct size was expressed as the percentage of the affected myocardial area (necrosis + inflammatory tissue) in all myocardial areas analyzed, with infarct area % = infarct area 100/total myocardial area. Immunostaining was used to evaluate the in vivo cardiomyogenesis and angiogenesis of the implants. The antibodies were used in a manner similar to the in vitro study. The number of neovessels, including sprouted capillaries, was counted per section, and a total of five sections per sample were analyzed. All of the slide analyses were performed using the ImageJ software.

All data are presented as the means SD. A one-way analysis of variance (ANOVA) with Tukeys test was used to verify statistically significant differences among groups via Origin Pro 8.5, with P < 0.05 being statistically significant (#, *P < 0.05; ##, **P < 0.01; ###, ***P < 0.001).

Acknowledgments: We would like to thank J. Zou and Y. Lin (IPSC core, NHLBI) for providing hiPSC-CMs and S. Anderson (Animal MRI core, NHLBI) for carrying out the MRI analysis. Funding: We also thank American Heart Association Transformative Project Award, NSF EBMS program grant #1856321, and NIH Directors New Innovator Award 1DP2EB020549-01 for financial support. Author contributions: H.C., C.L., Y.H., and L.G.Z. conceived the ideas and designed the experiments. H.C., X.Z., and S.-j.L. conducted the in vitro experiments. H.C., Y.H., C.L., Z.-x.Y., and H.S. carried out animal experiments. H.C., C.L., T.E., Y.H., Z.-x.Y., S.Y.H., M.B., M.M., J.P.F., and L.G.Z. performed data analysis and prepared the manuscript. Competing interests: A patent application describing the approach presented here was filed by H.C., L.G.Z., and Y.H. (US 62/571,684; PCT/US20 18/055707). The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional information related to this paper may be requested from the authors.

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4D physiologically adaptable cardiac patch: A 4-month in vivo study for the treatment of myocardial infarction - Science Advances

FDA Approves Merck’s KEYTRUDA (pembrolizumab) for the Treatment of Patients with Recurrent or Metastatic Cutaneous Squamous Cell Carcinoma (cSCC) that…

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck (NYSE: MRK), known as MSD outside the United States and Canada, announced today that the U.S. Food and Drug Administration (FDA) has approved KEYTRUDA, Mercks anti-PD-1 therapy, as monotherapy for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation. This approval is based on data from the Phase 2 KEYNOTE-629 trial, in which KEYTRUDA demonstrated meaningful efficacy and durability of response, with an objective response rate (ORR) of 34% (95% CI, 25-44), including a complete response rate of 4% and a partial response rate of 31%. Among responding patients, 69% had ongoing responses of six months or longer. After a median follow-up time of 9.5 months, the median duration of response (DOR) had not been reached (range, 2.7 to 13.1+ months).

Cutaneous squamous cell carcinoma is the second most common form of skin cancer, said Dr. Jonathan Cheng, vice president, clinical research, Merck Research Laboratories. In KEYNOTE-629, treatment with KEYTRUDA resulted in clinically meaningful and durable responses. Todays approval is great news for patients with cSCC and further demonstrates our commitment to bringing new treatment options to patients with advanced, difficult-to-treat cancers.

Immune-mediated adverse reactions, which may be severe or fatal, can occur with KEYTRUDA, including pneumonitis, colitis, hepatitis, endocrinopathies, nephritis and renal dysfunction, severe skin reactions, solid organ transplant rejection, and complications of allogeneic hematopoietic stem cell transplantation (HSCT). Based on the severity of the adverse reaction, KEYTRUDA should be withheld or discontinued and corticosteroids administered if appropriate. KEYTRUDA can also cause severe or life-threatening infusion-related reactions. Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. For more information, see Selected Important Safety Information below.

Data Supporting Approval

The efficacy of KEYTRUDA was investigated in patients with recurrent or metastatic cSCC enrolled in KEYNOTE-629 (NCT03284424), a multi-center, multi-cohort, non-randomized, open-label trial. The trial excluded patients with autoimmune disease or a medical condition that required immunosuppression. The major efficacy outcome measures were ORR and DOR as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, modified to follow a maximum of 10 target lesions and a maximum of five target lesions per organ.

Among the 105 patients treated, 87% received one or more prior lines of therapy and 74% received prior radiation therapy. Forty-five percent of patients had locally recurrent only cSCC, 24% had metastatic only cSCC and 31% had both locally recurrent and metastatic cSCC. The study population characteristics were: median age of 72 years (range, 29 to 95); 71% age 65 or older; 76% male; 71% White; 25% race unknown; 34% Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) of 0 and 66% ECOG PS of 1.

KEYTRUDA demonstrated an ORR of 34% (95% CI, 25-44) with a complete response rate of 4% and a partial response rate of 31%. Among the 36 responding patients, 69% had ongoing responses of six months or longer. After a median follow-up time of 9.5 months, the median DOR had not been reached (range, 2.7 to 13.1+ months).

Patients received KEYTRUDA 200 mg intravenously every three weeks until documented disease progression, unacceptable toxicity or a maximum of 24 months. Patients with initial radiographic disease progression could receive additional doses of KEYTRUDA during confirmation of progression unless disease progression was symptomatic, rapidly progressive, required urgent intervention, or occurred with a decline in performance status. Assessment of tumor status was performed every six weeks during the first year and every nine weeks during the second year.

Among the 105 patients with cSCC enrolled in KEYNOTE-629, the median duration of exposure to KEYTRUDA was 5.8 months (range, 1 day to 16.1 months). Patients with autoimmune disease or a medical condition that required systemic corticosteroids or other immunosuppressive medications were ineligible. Adverse reactions occurring in patients with cSCC were similar to those occurring in 2,799 patients with melanoma or non-small cell lung cancer (NSCLC) treated with KEYTRUDA as a single agent. Laboratory abnormalities (Grades 3-4) that occurred at a higher incidence included lymphopenia (11%).

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,200 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory classical Hodgkin lymphoma (cHL), or who have relapsed after 3 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 [combined positive score (CPS) 10], as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High (MSI-H) Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase (mut/Mb)] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options.

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Selected Important Safety Information for KEYTRUDA

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis, including fatal cases. Pneumonitis occurred in 3.4% (94/2799) of patients with various cancers receiving KEYTRUDA, including Grade 1 (0.8%), 2 (1.3%), 3 (0.9%), 4 (0.3%), and 5 (0.1%). Pneumonitis occurred in 8.2% (65/790) of NSCLC patients receiving KEYTRUDA as a single agent, including Grades 3-4 in 3.2% of patients, and occurred more frequently in patients with a history of prior thoracic radiation (17%) compared to those without (7.7%). Pneumonitis occurred in 6% (18/300) of HNSCC patients receiving KEYTRUDA as a single agent, including Grades 3-5 in 1.6% of patients, and occurred in 5.4% (15/276) of patients receiving KEYTRUDA in combination with platinum and FU as first-line therapy for advanced disease, including Grades 3-5 in 1.5% of patients.

Monitor patients for signs and symptoms of pneumonitis. Evaluate suspected pneumonitis with radiographic imaging. Administer corticosteroids for Grade 2 or greater pneumonitis. Withhold KEYTRUDA for Grade 2; permanently discontinue KEYTRUDA for Grade 3 or 4 or recurrent Grade 2 pneumonitis.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis. Colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 2 (0.4%), 3 (1.1%), and 4 (<0.1%). Monitor patients for signs and symptoms of colitis. Administer corticosteroids for Grade 2 or greater colitis. Withhold KEYTRUDA for Grade 2 or 3; permanently discontinue KEYTRUDA for Grade 4 colitis.

Immune-Mediated Hepatitis (KEYTRUDA) and Hepatotoxicity (KEYTRUDA in Combination With Axitinib)

Immune-Mediated Hepatitis

KEYTRUDA can cause immune-mediated hepatitis. Hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.4%), and 4 (<0.1%). Monitor patients for changes in liver function. Administer corticosteroids for Grade 2 or greater hepatitis and, based on severity of liver enzyme elevations, withhold or discontinue KEYTRUDA.

Hepatotoxicity in Combination With Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity with higher than expected frequencies of Grades 3 and 4 ALT and AST elevations compared to KEYTRUDA alone. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased ALT (20%) and increased AST (13%) were seen. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider more frequent monitoring of liver enzymes as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed.

Immune-Mediated Endocrinopathies

KEYTRUDA can cause adrenal insufficiency (primary and secondary), hypophysitis, thyroid disorders, and type 1 diabetes mellitus. Adrenal insufficiency occurred in 0.8% (22/2799) of patients, including Grade 2 (0.3%), 3 (0.3%), and 4 (<0.1%). Hypophysitis occurred in 0.6% (17/2799) of patients, including Grade 2 (0.2%), 3 (0.3%), and 4 (<0.1%). Hypothyroidism occurred in 8.5% (237/2799) of patients, including Grade 2 (6.2%) and 3 (0.1%). The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC (16%) receiving KEYTRUDA, as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. Hyperthyroidism occurred in 3.4% (96/2799) of patients, including Grade 2 (0.8%) and 3 (0.1%), and thyroiditis occurred in 0.6% (16/2799) of patients, including Grade 2 (0.3%). Type 1 diabetes mellitus, including diabetic ketoacidosis, occurred in 0.2% (6/2799) of patients.

Monitor patients for signs and symptoms of adrenal insufficiency, hypophysitis (including hypopituitarism), thyroid function (prior to and periodically during treatment), and hyperglycemia. For adrenal insufficiency or hypophysitis, administer corticosteroids and hormone replacement as clinically indicated. Withhold KEYTRUDA for Grade 2 adrenal insufficiency or hypophysitis and withhold or discontinue KEYTRUDA for Grade 3 or Grade 4 adrenal insufficiency or hypophysitis. Administer hormone replacement for hypothyroidism and manage hyperthyroidism with thionamides and beta-blockers as appropriate. Withhold or discontinue KEYTRUDA for Grade 3 or 4 hyperthyroidism. Administer insulin for type 1 diabetes, and withhold KEYTRUDA and administer antihyperglycemics in patients with severe hyperglycemia.

Immune-Mediated Nephritis and Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.1%), and 4 (<0.1%) nephritis. Nephritis occurred in 1.7% (7/405) of patients receiving KEYTRUDA in combination with pemetrexed and platinum chemotherapy. Monitor patients for changes in renal function. Administer corticosteroids for Grade 2 or greater nephritis. Withhold KEYTRUDA for Grade 2; permanently discontinue for Grade 3 or 4 nephritis.

Immune-Mediated Skin Reactions

Immune-mediated rashes, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) (some cases with fatal outcome), exfoliative dermatitis, and bullous pemphigoid, can occur. Monitor patients for suspected severe skin reactions and based on the severity of the adverse reaction, withhold or permanently discontinue KEYTRUDA and administer corticosteroids. For signs or symptoms of SJS or TEN, withhold KEYTRUDA and refer the patient for specialized care for assessment and treatment. If SJS or TEN is confirmed, permanently discontinue KEYTRUDA.

Other Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue in patients receiving KEYTRUDA and may also occur after discontinuation of treatment. For suspected immune-mediated adverse reactions, ensure adequate evaluation to confirm etiology or exclude other causes. Based on the severity of the adverse reaction, withhold KEYTRUDA and administer corticosteroids. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Based on limited data from clinical studies in patients whose immune-related adverse reactions could not be controlled with corticosteroid use, administration of other systemic immunosuppressants can be considered. Resume KEYTRUDA when the adverse reaction remains at Grade 1 or less following corticosteroid taper. Permanently discontinue KEYTRUDA for any Grade 3 immune-mediated adverse reaction that recurs and for any life-threatening immune-mediated adverse reaction.

The following clinically significant immune-mediated adverse reactions occurred in less than 1% (unless otherwise indicated) of 2799 patients: arthritis (1.5%), uveitis, myositis, Guillain-Barr syndrome, myasthenia gravis, vasculitis, pancreatitis, hemolytic anemia, sarcoidosis, and encephalitis. In addition, myelitis and myocarditis were reported in other clinical trials, including classical Hodgkin lymphoma, and postmarketing use.

Treatment with KEYTRUDA may increase the risk of rejection in solid organ transplant recipients. Consider the benefit of treatment vs the risk of possible organ rejection in these patients.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% (6/2799) of patients. Monitor patients for signs and symptoms of infusion-related reactions. For Grade 3 or 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Immune-mediated complications, including fatal events, occurred in patients who underwent allogeneic HSCT after treatment with KEYTRUDA. Of 23 patients with cHL who proceeded to allogeneic HSCT after KEYTRUDA, 6 (26%) developed graft-versus-host disease (GVHD) (1 fatal case) and 2 (9%) developed severe hepatic veno-occlusive disease (VOD) after reduced-intensity conditioning (1 fatal case). Cases of fatal hyperacute GVHD after allogeneic HSCT have also been reported in patients with lymphoma who received a PD-1 receptorblocking antibody before transplantation. Follow patients closely for early evidence of transplant-related complications such as hyperacute graft-versus-host disease (GVHD), Grade 3 to 4 acute GVHD, steroid-requiring febrile syndrome, hepatic veno-occlusive disease (VOD), and other immune-mediated adverse reactions.

In patients with a history of allogeneic HSCT, acute GVHD (including fatal GVHD) has been reported after treatment with KEYTRUDA. Patients who experienced GVHD after their transplant procedure may be at increased risk for GVHD after KEYTRUDA. Consider the benefit of KEYTRUDA vs the risk of GVHD in these patients.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with a PD-1 or PD-L1 blocking antibody in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-002, KEYTRUDA was permanently discontinued due to adverse reactions in 12% of 357 patients with advanced melanoma; the most common (1%) were general physical health deterioration (1%), asthenia (1%), dyspnea (1%), pneumonitis (1%), and generalized edema (1%). The most common adverse reactions were fatigue (43%), pruritus (28%), rash (24%), constipation (22%), nausea (22%), diarrhea (20%), and decreased appetite (20%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

In KEYNOTE-189, when KEYTRUDA was administered with pemetrexed and platinum chemotherapy in metastatic nonsquamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 20% of 405 patients. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonitis (3%) and acute kidney injury (2%). The most common adverse reactions (20%) with KEYTRUDA were nausea (56%), fatigue (56%), constipation (35%), diarrhea (31%), decreased appetite (28%), rash (25%), vomiting (24%), cough (21%), dyspnea (21%), and pyrexia (20%).

In KEYNOTE-407, when KEYTRUDA was administered with carboplatin and either paclitaxel or paclitaxel protein-bound in metastatic squamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 15% of 101 patients. The most frequent serious adverse reactions reported in at least 2% of patients were febrile neutropenia, pneumonia, and urinary tract infection. Adverse reactions observed in KEYNOTE-407 were similar to those observed in KEYNOTE-189 with the exception that increased incidences of alopecia (47% vs 36%) and peripheral neuropathy (31% vs 25%) were observed in the KEYTRUDA and chemotherapy arm compared to the placebo and chemotherapy arm in KEYNOTE-407.

In KEYNOTE-042, KEYTRUDA was discontinued due to adverse reactions in 19% of 636 patients with advanced NSCLC; the most common were pneumonitis (3%), death due to unknown cause (1.6%), and pneumonia (1.4%). The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia (7%), pneumonitis (3.9%), pulmonary embolism (2.4%), and pleural effusion (2.2%). The most common adverse reaction (20%) was fatigue (25%).

In KEYNOTE-010, KEYTRUDA monotherapy was discontinued due to adverse reactions in 8% of 682 patients with metastatic NSCLC; the most common was pneumonitis (1.8%). The most common adverse reactions (20%) were decreased appetite (25%), fatigue (25%), dyspnea (23%), and nausea (20%).

Adverse reactions occurring in patients with SCLC were similar to those occurring in patients with other solid tumors who received KEYTRUDA as a single agent.

In KEYNOTE-048, KEYTRUDA monotherapy was discontinued due to adverse events in 12% of 300 patients with HNSCC; the most common adverse reactions leading to permanent discontinuation were sepsis (1.7%) and pneumonia (1.3%). The most common adverse reactions (20%) were fatigue (33%), constipation (20%), and rash (20%).

In KEYNOTE-048, when KEYTRUDA was administered in combination with platinum (cisplatin or carboplatin) and FU chemotherapy, KEYTRUDA was discontinued due to adverse reactions in 16% of 276 patients with HNSCC. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonia (2.5%), pneumonitis (1.8%), and septic shock (1.4%). The most common adverse reactions (20%) were nausea (51%), fatigue (49%), constipation (37%), vomiting (32%), mucosal inflammation (31%), diarrhea (29%), decreased appetite (29%), stomatitis (26%), and cough (22%).

In KEYNOTE-012, KEYTRUDA was discontinued due to adverse reactions in 17% of 192 patients with HNSCC. Serious adverse reactions occurred in 45% of patients. The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia, dyspnea, confusional state, vomiting, pleural effusion, and respiratory failure. The most common adverse reactions (20%) were fatigue, decreased appetite, and dyspnea. Adverse reactions occurring in patients with HNSCC were generally similar to those occurring in patients with melanoma or NSCLC who received KEYTRUDA as a monotherapy, with the exception of increased incidences of facial edema and new or worsening hypothyroidism.

In KEYNOTE-087, KEYTRUDA was discontinued due to adverse reactions in 5% of 210 patients with cHL. Serious adverse reactions occurred in 16% of patients; those 1% included pneumonia, pneumonitis, pyrexia, dyspnea, GVHD, and herpes zoster. Two patients died from causes other than disease progression; 1 from GVHD after subsequent allogeneic HSCT and 1 from septic shock. The most common adverse reactions (20%) were fatigue (26%), pyrexia (24%), cough (24%), musculoskeletal pain (21%), diarrhea (20%), and rash (20%).

In KEYNOTE-170, KEYTRUDA was discontinued due to adverse reactions in 8% of 53 patients with PMBCL. Serious adverse reactions occurred in 26% of patients and included arrhythmia (4%), cardiac tamponade (2%), myocardial infarction (2%), pericardial effusion (2%), and pericarditis (2%). Six (11%) patients died within 30 days of start of treatment. The most common adverse reactions (20%) were musculoskeletal pain (30%), upper respiratory tract infection and pyrexia (28% each), cough (26%), fatigue (23%), and dyspnea (21%).

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FDA Approves Merck's KEYTRUDA (pembrolizumab) for the Treatment of Patients with Recurrent or Metastatic Cutaneous Squamous Cell Carcinoma (cSCC) that...

Over $8M in 2020 Stem Cell Funding Awards Continue to Fuel Marylands Leading Cell Therapy Industry – BioBuzz

The Maryland Stem Cell Research Commission (The Commission) recently announced over $7M in Maryland Stem Cell Fund (MSCF) grant awards for its second round of 2020 MSCF fund recipients. The MSCF, which is a program of the Maryland Technology Development Corporation (TEDCO), has awarded $157M in funding to BioHealth Capital Region (BHCR) companies seeking to accelerate stem cell research, therapies and commercialization of products since 2007.

The $7M in new funding follows MSCFs announcement in September 2019 of over $1.3M in grants for the first cohort of 2020 recipients, bringing the total 2020 MSCF award tally to approximately $8.3M for the year. The financial awards are delivered across a wide range of areas, including clinical, commercialization, validation, launch, discovery, and post-doctoral fellowships. The first cohort of funding included three commercialization and two validation awards; the second, larger recipient pool included one clinical, one commercialization, one validation, four launches, 11 discovery, and five post-doctoral awards.

Notable BHCR MSCF recipients included:

Dr. Luis Garza of Johns Hopkins University (JHU) received a clinical grant to support clinical trials for his autologous volar fibroblast injection into the stump site of amputees. The trials are exploring ways to make the skin where a prosthetic limb meets the stump site tougher and less irritable to the wearer. Skin irritation is a major issue for those with prosthetic limbs and is often a cause for individuals to stop wearing their prosthesis.

Vita Therapeutics, a company that spun out of JHU, was awarded a 300K MSCF grant to support the commercialization of the companys satellite stem cell therapy for limb-girdle Muscular Dystrophy. According to the National Organization for Rare Disorders (NORD), Limb-girdle muscular dystrophies (LGMD) are a group of rare progressive genetic disorders that are characterized by wasting (atrophy) and weakness of the voluntary muscles of the hip and shoulder areas (limb-girdle area). Vita Therapeutics is led by CEO Douglass Falk, who is a JHU alum.

Jamie Niland, VP of Baltimore, Marylands Neoprogen Inc. received part of $892,080K in funding that was part of MSCFs first 2020 grant round. Jamie is the son of Bill Niland, Neoprogens current CEO and the former leader of Baltimore, Maryland life science community anchor Harpoon Medical, which was acquired by Edwards Scientific in 2017. The award was for Neoprogens neonatal cardiac stem cells for the heart tissue regeneration program.

Dr. Brian Pollok of Rockville, Marylands Propagenix, Inc., was also the recipient of a commercialization award for his Apical Surface-Outward (ASO) airway organoids, which is a potential novel cell system for drug discovery and personalized medicine. Propagenix develops innovative new technologies that address unmet needs in epithelial cell biologyfor applications in life science research as well as in precision diagnostics, and next-generation therapeutics such as immune-oncology, tissue engineering, and regenerative medicine, according to the companys website.

In addition, Dr. Ines Silva, R&D Manager of REPROCELL, USA received an MSCF commercialization grant for its work on building a commercial neural cell bank from patient-derived induced pluripotent stem cells. REPROCELL was founded in Japan in 2003 and acquired BioServe in Beltsville, Maryland in 2014.

Dr. Sashank Reddy, the founder of JHU startup LifeSprout and Medical Director, Johns Hopkins Technology Ventures Johns Hopkins University, received a portion of the $1,334,462 distributed for launch grants in 2020. The grant will go to support the launch of regenerative cell therapies for soft tissue restoration. LifeSprout recently closed a $28.5M seed round.

Past MSCF grant recipients include Frederick, Marylands RoosterBio, Inc. and Theradaptive, Inc., and Baltimore, Marylands Gemstone Biotherapeutics and Domicell, Inc., among others.

TEDCOs MSRF program continues to lend its deep support and ample funding to build and grow Marylands burgeoning and exciting regenerative medicine industry. Well be keeping a close eye on these companies as they grow and make future contributions to the thriving BHCR biocluster.

Steve has over 20 years experience in copywriting, developing brand messaging and creating marketing strategies across a wide range of industries, including the biopharmaceutical, senior living, commercial real estate, IT and renewable energy sectors, among others. He is currently the Principal/Owner of StoryCore, a Frederick, Maryland-based content creation and execution consultancy focused on telling the unique stories of Maryland organizations.

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Over $8M in 2020 Stem Cell Funding Awards Continue to Fuel Marylands Leading Cell Therapy Industry - BioBuzz

Insight on the Growth of Autologous Stem Cell Based Therapies Market Growth with Challenges, Standardization, Competitive Market Share and Top Players…

The Autologous Stem Cell Based Therapies Market globally is a standout amongst the most emergent and astoundingly approved sectors. This worldwide market has been developing at a higher pace with the development of imaginative frameworks and a developing end-client tendency.

Autologous Stem Cell Based Therapies market reports deliver insight and expert analysis into key consumer trends and behaviour in marketplace, in addition to an overview of the market data and key brands. Autologous Stem Cell Based Therapies market reports provides all data with easily digestible information to guide every businessmans future innovation and move business forward.

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The worldwide Autologous Stem Cell Based Therapies market is an enlarging field for top market players,

The key players covered in this studyRegeneusMesoblastPluristem Therapeutics IncU.S. STEM CELL, INC.Brainstorm Cell TherapeuticsTigenixMed cell Europe

Market segment by Type, the product can be split intoEmbryonic Stem CellResident Cardiac Stem CellsUmbilical Cord Blood Stem Cells

Market segment by Application, split intoNeurodegenerative DisordersAutoimmune DiseasesCardiovascular Diseases

Market segment by Regions/Countries, this report coversUnited StatesEuropeChinaJapanSoutheast AsiaIndiaCentral & South America

The study objectives of this report are:To analyze global Autologous Stem Cell Based Therapies status, future forecast, growth opportunity, key market and key players.To present the Autologous Stem Cell Based Therapies development in United States, Europe and China.To strategically profile the key players and comprehensively analyze their development plan and strategies.To define, describe and forecast the market by product type, market and key regions.

In this study, the years considered to estimate the market size of Autologous Stem Cell Based Therapies are as follows:History Year: 2014-2018Base Year: 2018Estimated Year: 2019Forecast Year 2019 to 2025For the data information by region, company, type and application, 2018 is considered as the base year. Whenever data information was unavailable for the base year, the prior year has been considered.

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This Autologous Stem Cell Based Therapies report begins with a basic overview of the market. The analysis highlights the opportunity and Autologous Stem Cell Based Therapies industry trends that are impacted the market that is global. Players around various regions and analysis of each industry dimensions are covered under this report. The analysis also contains a crucial Autologous Stem Cell Based Therapies insight regarding the things which are driving and affecting the earnings of the market. The Autologous Stem Cell Based Therapies report comprises sections together side landscape which clarifies actions such as venture and acquisitions and mergers.

The Report offers SWOT examination and venture return investigation, and other aspects such as the principle locale, economic situations with benefit, generation, request, limit, supply, and market development rate and figure.

Quantifiable data:-

Geographically, this report studies the top producers and consumers, focuses on product capacity, production, value, consumption, market share and growth opportunity in these key regions, covering North America, Europe, China, Japan, Southeast Asia, India

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Research objectives and Reason to procure this report:-

Finally, the global Autologous Stem Cell Based Therapies market provides a total research decision and also sector feasibility of investment in new projects will be assessed. Autologous Stem Cell Based Therapies industry is a source of means and guidance for organizations and individuals interested in their market earnings.

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Insight on the Growth of Autologous Stem Cell Based Therapies Market Growth with Challenges, Standardization, Competitive Market Share and Top Players...

Trending: Autologous Stem Cell Based Therapies 2020: Global Size, Supply-Demand, Product Type and End User Analysis To 2026 – Weekly Wall

LOS ANGELES, United States: QY Research has recently published a report, titled Global Autologous Stem Cell Based Therapies Market Size, Status and Forecast 2020-2026. The market research report is a brilliant, complete, and much-needed resource for companies, stakeholders, and investors interested in the global Autologous Stem Cell Based Therapies market. It informs readers about key trends and opportunities in the global Autologous Stem Cell Based Therapies market along with critical market dynamics expected to impact the global market growth. It offers a range of market analysis studies, including production and consumption, sales, industry value chain, competitive landscape, regional growth, and price. On the whole, it comes out as an intelligent resource that companies can use to gain a competitive advantage in the global Autologous Stem Cell Based Therapies market.

Key companies operating in the global Autologous Stem Cell Based Therapies market include , Regeneus, Mesoblast, Pluristem Therapeutics Inc, US STEM CELL, INC., Brainstorm Cell Therapeutics, Tigenix, Med cell Europe, Autologous Stem Cell Based Therapies

Get PDF Sample Copy of the Report to understand the structure of the complete report: (Including Full TOC, List of Tables & Figures, Chart) :

https://www.qyresearch.com/sample-form/form/1889061/global-autologous-stem-cell-based-therapies-market

Segmental Analysis

Both developed and emerging regions are deeply studied by the authors of the report. The regional analysis section of the report offers a comprehensive analysis of the global Autologous Stem Cell Based Therapies market on the basis of region. Each region is exhaustively researched about so that players can use the analysis to tap into unexplored markets and plan powerful strategies to gain a foothold in lucrative markets.

Global Autologous Stem Cell Based Therapies Market Segment By Type:

, Embryonic Stem Cell, Resident Cardiac Stem Cells, Umbilical Cord Blood Stem Cells Autologous Stem Cell Based Therapies

Global Autologous Stem Cell Based Therapies Market Segment By Application:

, Neurodegenerative Disorders, Autoimmune Diseases, Cardiovascular Diseases

Competitive Landscape

Competitor analysis is one of the best sections of the report that compares the progress of leading players based on crucial parameters, including market share, new developments, global reach, local competition, price, and production. From the nature of competition to future changes in the vendor landscape, the report provides in-depth analysis of the competition in the global Autologous Stem Cell Based Therapies market.

Key companies operating in the global Autologous Stem Cell Based Therapies market include , Regeneus, Mesoblast, Pluristem Therapeutics Inc, US STEM CELL, INC., Brainstorm Cell Therapeutics, Tigenix, Med cell Europe, Autologous Stem Cell Based Therapies

Key questions answered in the report:

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TOC

1 Report Overview1.1 Study Scope1.2 Key Market Segments1.3 Players Covered: Ranking by Autologous Stem Cell Based Therapies Revenue1.4 Market by Type1.4.1 Global Autologous Stem Cell Based Therapies Market Size Growth Rate by Type: 2020 VS 20261.4.2 Embryonic Stem Cell1.4.3 Resident Cardiac Stem Cells1.4.4 Umbilical Cord Blood Stem Cells1.5 Market by Application1.5.1 Global Autologous Stem Cell Based Therapies Market Share by Application: 2020 VS 20261.5.2 Neurodegenerative Disorders1.5.3 Autoimmune Diseases1.5.4 Cardiovascular Diseases1.6 Study Objectives1.7 Years Considered 2 Global Growth Trends2.1 Global Autologous Stem Cell Based Therapies Market Perspective (2015-2026)2.2 Global Autologous Stem Cell Based Therapies Growth Trends by Regions2.2.1 Autologous Stem Cell Based Therapies Market Size by Regions: 2015 VS 2020 VS 20262.2.2 Autologous Stem Cell Based Therapies Historic Market Share by Regions (2015-2020)2.2.3 Autologous Stem Cell Based Therapies Forecasted Market Size by Regions (2021-2026)2.3 Industry Trends and Growth Strategy2.3.1 Market Top Trends2.3.2 Market Drivers2.3.3 Market Challenges2.3.4 Porters Five Forces Analysis2.3.5 Autologous Stem Cell Based Therapies Market Growth Strategy2.3.6 Primary Interviews with Key Autologous Stem Cell Based Therapies Players (Opinion Leaders) 3 Competition Landscape by Key Players3.1 Global Top Autologous Stem Cell Based Therapies Players by Market Size3.1.1 Global Top Autologous Stem Cell Based Therapies Players by Revenue (2015-2020)3.1.2 Global Autologous Stem Cell Based Therapies Revenue Market Share by Players (2015-2020)3.1.3 Global Autologous Stem Cell Based Therapies Market Share by Company Type (Tier 1, Tier 2 and Tier 3)3.2 Global Autologous Stem Cell Based Therapies Market Concentration Ratio3.2.1 Global Autologous Stem Cell Based Therapies Market Concentration Ratio (CR5 and HHI)3.2.2 Global Top 10 and Top 5 Companies by Autologous Stem Cell Based Therapies Revenue in 20193.3 Autologous Stem Cell Based Therapies Key Players Head office and Area Served3.4 Key Players Autologous Stem Cell Based Therapies Product Solution and Service3.5 Date of Enter into Autologous Stem Cell Based Therapies Market3.6 Mergers & Acquisitions, Expansion Plans 4 Market Size by Type (2015-2026)4.1 Global Autologous Stem Cell Based Therapies Historic Market Size by Type (2015-2020)4.2 Global Autologous Stem Cell Based Therapies Forecasted Market Size by Type (2021-2026) 5 Market Size by Application (2015-2026)5.1 Global Autologous Stem Cell Based Therapies Market Size by Application (2015-2020)5.2 Global Autologous Stem Cell Based Therapies Forecasted Market Size by Application (2021-2026) 6 North America6.1 North America Autologous Stem Cell Based Therapies Market Size (2015-2020)6.2 Autologous Stem Cell Based Therapies Key Players in North America (2019-2020)6.3 North America Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)6.4 North America Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 7 Europe7.1 Europe Autologous Stem Cell Based Therapies Market Size (2015-2020)7.2 Autologous Stem Cell Based Therapies Key Players in Europe (2019-2020)7.3 Europe Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)7.4 Europe Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 8 China8.1 China Autologous Stem Cell Based Therapies Market Size (2015-2020)8.2 Autologous Stem Cell Based Therapies Key Players in China (2019-2020)8.3 China Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)8.4 China Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 9 Japan9.1 Japan Autologous Stem Cell Based Therapies Market Size (2015-2020)9.2 Autologous Stem Cell Based Therapies Key Players in Japan (2019-2020)9.3 Japan Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)9.4 Japan Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 10 Southeast Asia10.1 Southeast Asia Autologous Stem Cell Based Therapies Market Size (2015-2020)10.2 Autologous Stem Cell Based Therapies Key Players in Southeast Asia (2019-2020)10.3 Southeast Asia Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)10.4 Southeast Asia Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 11 India11.1 India Autologous Stem Cell Based Therapies Market Size (2015-2020)11.2 Autologous Stem Cell Based Therapies Key Players in India (2019-2020)11.3 India Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)11.4 India Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 12 Central & South America12.1 Central & South America Autologous Stem Cell Based Therapies Market Size (2015-2020)12.2 Autologous Stem Cell Based Therapies Key Players in Central & South America (2019-2020)12.3 Central & South America Autologous Stem Cell Based Therapies Market Size by Type (2015-2020)12.4 Central & South America Autologous Stem Cell Based Therapies Market Size by Application (2015-2020) 13 Key Players Profiles13.1 Regeneus13.1.1 Regeneus Company Details13.1.2 Regeneus Business Overview13.1.3 Regeneus Autologous Stem Cell Based Therapies Introduction13.1.4 Regeneus Revenue in Autologous Stem Cell Based Therapies Business (2015-2020))13.1.5 Regeneus Recent Development13.2 Mesoblast13.2.1 Mesoblast Company Details13.2.2 Mesoblast Business Overview13.2.3 Mesoblast Autologous Stem Cell Based Therapies Introduction13.2.4 Mesoblast Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.2.5 Mesoblast Recent Development13.3 Pluristem Therapeutics Inc13.3.1 Pluristem Therapeutics Inc Company Details13.3.2 Pluristem Therapeutics Inc Business Overview13.3.3 Pluristem Therapeutics Inc Autologous Stem Cell Based Therapies Introduction13.3.4 Pluristem Therapeutics Inc Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.3.5 Pluristem Therapeutics Inc Recent Development13.4 US STEM CELL, INC.13.4.1 US STEM CELL, INC. Company Details13.4.2 US STEM CELL, INC. Business Overview13.4.3 US STEM CELL, INC. Autologous Stem Cell Based Therapies Introduction13.4.4 US STEM CELL, INC. Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.4.5 US STEM CELL, INC. Recent Development13.5 Brainstorm Cell Therapeutics13.5.1 Brainstorm Cell Therapeutics Company Details13.5.2 Brainstorm Cell Therapeutics Business Overview13.5.3 Brainstorm Cell Therapeutics Autologous Stem Cell Based Therapies Introduction13.5.4 Brainstorm Cell Therapeutics Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.5.5 Brainstorm Cell Therapeutics Recent Development13.6 Tigenix13.6.1 Tigenix Company Details13.6.2 Tigenix Business Overview13.6.3 Tigenix Autologous Stem Cell Based Therapies Introduction13.6.4 Tigenix Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.6.5 Tigenix Recent Development13.7 Med cell Europe13.7.1 Med cell Europe Company Details13.7.2 Med cell Europe Business Overview13.7.3 Med cell Europe Autologous Stem Cell Based Therapies Introduction13.7.4 Med cell Europe Revenue in Autologous Stem Cell Based Therapies Business (2015-2020)13.7.5 Med cell Europe Recent Development 14 Analysts Viewpoints/Conclusions 15 Appendix15.1 Research Methodology15.1.1 Methodology/Research Approach15.1.2 Data Source15.2 Disclaimer15.3 Author Details

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Trending: Autologous Stem Cell Based Therapies 2020: Global Size, Supply-Demand, Product Type and End User Analysis To 2026 - Weekly Wall

Microneedle-mediated gene delivery for the treatment of ischemic myocardial disease – Science Advances

Abstract

Cardiovascular disorders are still the primary cause of mortality worldwide. Although intramyocardial injection can effectively deliver agents to the myocardium, this approach is limited because of its restriction to needle-mediated injection and the minor retention of agents in the myocardium. Here, we engineered phase-transition microneedles (MNs) coated with adeno-associated virus (AAV) and achieved homogeneous distribution of AAV delivery. Bioluminescence imaging revealed the successful delivery and transfection of AAV-luciferase. AAVgreen fluorescent proteintransfected cardiomyocytes were homogeneously distributed on postoperative day 28. AAVvascular endothelial growth factor (VEGF)loaded MNs improved heart function by enhancing VEGF expression, promoting functional angiogenesis, and activating the Akt signaling pathway. The results indicated the superiority of MNs over direct muscle injection. Consequently, MNs might emerge as a promising tool with great versatility for delivering various agents to treat ischemic myocardial disease.

The American Heart Association has stated that cardiovascular disease (CVD) is the primary cause of mortality worldwide, leading to more than 17.3 million deaths per year; the number of deaths is estimated to exceed 23.6 million by 2030 (1). Thus, all potential treatment strategies to preserve left ventricle (LV) function by limiting infarct expansion and alleviating adverse remodeling are currently being investigated (2). To date, various injectable agents, including biomaterials, cells, genes, and proteins (37), have been studied and shown to have various advantages. Direct intramyocardial delivery of agents through myocardial transfection in the ischemic regions where vascular delivery procedures were excluded and the systemic administration of vectors might pose potential hazards following the procedures of myocardial revascularization was suitable (8). However, the effects of intramyocardially delivered therapeutics are restricted to the site of injection (911). Another major drawback is the minor myocardial retention of injected agents. Previous reports have demonstrated that few injected cells are retained in injured hearts, which is one of the principal reasons for the failure of cell therapy for myocardial repair (12, 13). On the other hand, all body tissues can be exposed to drugs if they accidently enter the left ventricular cavity, which can reduce therapeutic efficacy and contribute to unexpected results. Therefore, the current limitations associated with this strategy must be mitigated. Cardiac gene transfer has been considered to be a promising therapeutic tool in the field of cardiology (14, 15). Adeno-associated virus (AAV)9, a serotype with high cardiac tropism, persistent transgene expression, and low pathogenicity, has also been applied for cardiac gene therapy (16). Transgenic expression of AAVs starts from 5 to 7 days after administration, and remarkably elevated viral transfection efficiency is achieved at weeks 2 to 3. Delivered vectors continue to express their transgenes for 6 to 12 months in vivo (17, 18). AAV-mediated gene expression in vivo declines with time due to promoter shutoff and loss of AAV-transduced cells and AAV particles (19).

Microneedles (MNs) are an array of small needles, up to 1 mm in length, that provide secure channels for the passage of therapeutic substances (2, 20), especially macromolecules, without causing skin injury or pain; these macromolecules include nucleic acids in the form of genes (21), vaccines (22), and proteins (2325). The precise and efficient transfusion and homogeneous distribution of therapeutic agents delivered via MNs make MN-mediated delivery a promising new administration method for ischemic heart disease (IHD) treatment. In this study, we fabricated phase-transition MNs (PTMs) and studied their properties as well as their safety and practicality for experimental application. A schematic illustrating the overall study design using AAV-harboring MNs (MN-AAV) is shown in Fig. 1A. Figure 1B represents our practice for the application of MNs to deliver therapeutic agents via endoscopy assisted microthoracotomy surgery. A series of endoscopic images demonstrate the in vivo application of MNs to deliver therapeutic agents to the rat heart as shown in Fig. 1C. MNs loaded with fluorescent fluorescein isothiocyanate (FITC)labeled AAV (MN-FITC-AAV) and AAV containing the luciferase coding sequence (MN-AAV-LUC) enabled successful therapeutic agent delivery and gene transfection of target heart regions. AAVgreen fluorescent protein (GFP)loaded MNs (MN-AAV-GFP) enabled fine distribution of AAV particles, presenting an advantage over direct injection (DI), after which positive cells were limited in location to the injection site in vivo. Thus, MN-AAV, which allow agents to be burst-released, can achieve even distributions of agents at the target myocardium rather than confining the agents to the site of administration. Heart performance and histological examinations showed that MNs loaded with AAV vectorencoding vascular endothelial growth factor gene (MN-AAV-VEGF) could improve cardiac function, reduce scar size, ameliorate adverse remodeling, and elevate myocardial perfusion in a rat model of myocardial infarction (MI). MN-mediated gene therapy showed distinct superiority over DI and may therefore provide an alternative, minimally invasive therapeutic option for heart diseases.

(A) Ischemic hearts were administered MN-AAV with the assistance of a customized apparatus. The MNs swelled following application; consequently, the therapeutic agents were burst-released into precise regions to ameliorate cardiac dysfunction through angiogenic effects. (B) Diagram of our practice for the application of the MNs to rat heart via endoscopy assisted microthoracotomy surgery. (C) A series of endoscopic images demonstrating the application of MNs for delivery of therapeutic agents to a rat heart. Scale bars, 600 m. Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

The fabrication process of the MN patches is shown schematically in fig. S1. The prototype MN-AAV patch was 6 mm in diameter and contained 44.75 1.28 needle tips with base widths of 334 22.88 m, spacing of 465.3 39.51 m, and heights of 850 3.25 m as shown in the scanning electron microscopy (SEM) image (Fig. 2A). The representative stress-strain curves are shown in Fig. 2B (left). Uniaxial tensile tests showed that the MNs had a Youngs modulus of 13.13 1.34 MPa, while the MN-AAV had a Youngs modulus of 12.28 0.80 MPa. The Youngs modulus of the MN group was higher than that of the MN-AAV group; however, this difference was not significant (P > 0.05) (Fig. 2B). Both the MN and MN-AAV had higher moduli than the native myocardium (modulus, several tens of kilopascal), indicating that the stiffness of the MNs with or without AAV loading was sufficient to penetrate the soft myocardium.

(A) SEM images of MNs. (B) Representative stress-strain curves between the group of MNs with AAV (MN-AAV) or MNs without AAV. The histograms represent the comparison test of the two groups. n = 4 patches in each group. (C) Transitions between the dried and swollen states of the MNs. The histograms show the fold changes in MN volume between the dried and swollen stages (n = 8 MN tips, randomly selected from three patches). Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. (D) Release kinetics of MN-AAV. (E) Fluorescent images (scale bars, 500 m) and magnified images (scale bars, 250 m) indicating MNs surface-coated with FITC-AAV (green) particles and MNs without loading of FITC-AAV. (F) The three-dimensional (3D) construction images of MN-FITC-AAV. All data are reported as the means SD. NS, not significant.

The swelling capacity of the MN bodies was monitored, and volume expansion was measured and calculated using a previously published method. The mean base diameter of the swollen MN bodies among three patches was 670.5 81.63 m, 678.9 89.17 m, and 683.4 67.31 m. The mean height of the swollen MN bodies was 1704 56.75 m, 1701 73 m, and 1705 66.63 m. Measurements of total recorded MN tips in height and base diameter were 1704 64.89 m and 677.6 78.93 m, respectively, which were significantly greater than those of the MN bodies in the initial state. The transitions between the dried and swollen states of MNs are shown in Fig. 2C. Swelling led to an 8.3-fold variation, indicating that the MN tips exhibited a high swelling ratio.

Quantification of AAV release into supernatant collected after incubation at predetermined time points indicated a burst release model. A schematic of the experimental procedures performed to collect the released AAV fluid is provided in fig. S2. As shown in Fig. 2D, burst release led to increased initial AAV delivery, as follows: 90.93% of the virus was released in a 2-s period, while 92.42% of the virus was released in a 5-s period, and slower release followed the initial rapid release. Almost complete release was achieved by 24 hours. The titers of elution fluid released from MN-AAV (n = 3) were determined by real-time polymerase chain reaction (PCR), which indicated that 4.93 1010 vector genomes (vg) of AAV were loaded in each MN array. We also loaded greater amounts of AAV (the quantity of loaded AAV achieved 1011 vg) with three gradient quantities (1, 1.5, and 2; n = 4 patches in each group). The amounts of loaded AAV were calculated to be 3.14 1011 vg, 5.04 1011 vg, and 6.03 1011 vg, respectively; the fold differences of these groups were 1-, 1.69-, and 2.02-folds, respectively. Consequently, we could control the quantity of loaded vectors in each MN array by varying the amount of AAV solutions added. Furthermore, MNs exhibited outstanding drug-loading capacity.

To confirm AAV binding, we performed a critical examination of virus labeling with FITC dyes. As expected, compared with the control MNs without FITC-AAV, the AAV-loaded MNs revealed a strong fluorescence signal on the surface of the MN bodies (Fig. 2E, left and middle). Conversely, a specific fluorescence signal was absent in the control MN group (Fig. 2E, right). Ortho view (fig. S3) of a confocal laser scanning micrograph of z-stack images visualizes the MN tip as transverse section (x-y) and lateral section (x-z and y-z) views. The three-dimensional (3D) images generated by confocal microscopy confirmed that FITC-labeled AAV was successfully coated on the surface of the MN bodies (Fig. 2F). The fluorescence intensity of the fluorescent images acquired by confocal microscopy at the middle of MN bodies (400 m from the base) was measured and compared among 11 randomly selected MNs in one patch. The average optical was measured using Image-Pro Plus software to evaluate the fluorescence intensity. The fluorescence intensity was 0.1127 0.0233 with a little variation among the MNs. In addition, the intensity analysis at the middle of MN bodies among three MN patches (8 or 11 MNs were randomly selected from each patch) was measured and compared. No differences were observed among three patches (0.1127 0.0233 versus 0.1156 0.0254 versus 0.1084 0.0279, all P > 0.05 in the multigroup comparisons) (fig. S4A).

A schematic of the cell culture procedures is provided in Fig. 3A. The released vectors were tested for their infectious capacity and transgene expression in human embryonic kidney (HEK) 293 cells by flow cytometry and fluorescence microscopy. After a 3-day incubation, the distribution of GFP-positive cells was determined using fluorescence microscopy (Fig. 3B). Flow cytometry analysis revealed that 5.14% of the cells were transduced by the supernatant released from MN-AAV2-GFP (Fig. 3C). A comparison analysis of the GFP-positive cells indicated that the percentage of positive cells was significantly different between the MN-AAV2-GFP and normal control (NC) groups (P = 0.0045). We evaluated the efficiency of AAV9 transduction into HEK 293 cells between virus-containing MNs subjected to a freeze-thaw process (MN-AAV-FT) and those not subjected to a freeze-thaw process (MN-AAV-NFT). There was no difference between the MN-AAV-FT and MN-AAV-NFT groups (the relative percentage of the transduction efficiency was 97.2% versus 100%) (fig. S5).

(A) Schematic of the cell culture experimental procedures performed to investigate the cell infectivity of released AAV. (B) Representative fluorescent images of GFP-positive cells in the MN-AAV-GFP group captured under a confocal microscope. Scale bars, 100 m. DAPI, 4,6-diamidino-2-phenylindole. (C) Qualification and comparison of GFP-positive cells between normal 293 cells and AAV-GFP transfected cells as detected by flow cytometry. SSC-A, side-scatter area; FSC-A, forward-scatter area. (D) Representative images of crystal violetstained migratory human umbilical vein endothelial cells (HUVECs) on the porous membranes of Transwell inserts among the three groups and histograms of the numbers of migrated cells. Five random fields were selected for the statistical analysis. All data are reported as the means SD. **P < 0.01 and ****P < 0.0001.

The angiogenic effect of AAV-VEGF was evaluated in vitro. Endothelial cell migration is of great importance in neovessel formation; therefore, the influence of the AAV-VEGFtransfected H9C2 cell culture supernatant on human umbilical vein endothelial cell (HUVEC) migration was assessed. The assay indirectly proved that the H9C2 cells infected by MN-AAV-VEGF released vectors could secrete VEGF into the culture supernatant, which strongly stimulated HUVEC migration [179.8 6.76 per high-power field (HPF) in the H9C2-VEGF group] compared to that observed in the AAV-GFPinfected H9C2 cells (79.2 8.53 per HPF in the H9C2-GFP group, P < 0.0001) and the NC H9C2 group (80.8 9.34 per HPF, P < 0.0001; Fig. 3D).

Figure 4A briefly illustrates the procedures used to demonstrate successful AAV delivery and gene transfection mediated by MN-AAV in vivo. The precise region of the rat heart that received the methylene blueloaded MNs was imaged and dissected. A customized vacuum apparatus was used for the implantation of MN patch (Fig. 1A and movies S1, S2, and S3). As shown in Fig. 4B and movies S3 and S4, the epicardium with puncture spots and the myocardium were stained by the released dyes. Similarly, MN- FITC-AAV were used to further confirm the successful insertion of the MNs. Fluorescent images of the horizontal and vertical sections of the LV wall indicated the penetration of the MNs, which resulted in an even distribution of agents (Fig. 4C, middle) compared to the distribution in the DI group (Fig. 4C, left). The fluorescence intensity in horizontal sections of rat hearts (Fig. 4C, middle) after MN-FITC-AAV administration (n = 3 animals; 15 puncture points were analyzed in each fluorescent image) was measured, and no significant differences were observed (0.1324 0.0172 versus 0.1289 0.0207 versus 0.1337 0.0212, all P > 0.05 in the multigroup comparisons), confirming the uniformity of AAV loading (fig. S4B). In the transverse plane of the LV following MN-FITC-AAV application (Fig. 4C, right), the penetration depth of MNs into the LV wall was approximately 1000 m.

(A) Schematic illustrating the study design, involving the MN application in this section. (B) Confirmed insertion of methylene blueloaded MNs into the myocardium. The black arrow denotes an area of methylene bluestained myocardium. Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. (C) Representative fluorescent and hematoxylin and eosin (HE) images of LV walls that received DI of FITC-AAV and MN-FITC-AAV. The LV wall was cryosectioned horizontally (n = 3 animals per group; scale bars, 500 m) or transversely (scale bars, 400 m) for the MN-FITC-AAVtreated hearts. The dashed line denotes the shape of MN-FITC-AAV following application. (D) Representative echocardiographic images and left ventricular function parameters between the MN and NC groups. The data are presented as the means SD; n = 3 animals per group. (E) Representative images of bioluminescence (n = 5 animals per group) and Western blot (WB) assay results (n = 3 animals per group) 4 weeks following MN application. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Representative fluorescence micrographs showing the spatial distribution of GFP-positive cells (green) in the MN-AAV-GFP and DI-AAV-GFP groups at day 28. Cardiomyocytes were identified by anti-cTnT (cardiac troponin T) antibodies (red); nuclei were stained with DAPI (blue). n = 5 animals per group. Scale bars, 200 m. Separated and merged distribution data of fluorescent signals between the MN-AAV-GFP and DI-AAV-GFP groups are presented. All data are reported as the means SD.

Inflammatory staining was performed on sections from hearts subjected to MN-AAV and DI-AAV treatments to reveal signs of tissue inflammation. Normal rats were used as controls. We quantified CD68-positive inflammatory cell infiltration and found that the tissue densities of CD68-positive cells were indistinguishable among the three groups (P > 0.05) (fig. S6). In addition, we examined the heart performance of rats that underwent MN administration, which also confirmed the safety of the MN patch. The echocardiographic results indicated that MN application did not induce any functional impairment. Ejection fraction (EF), fractional shortening (FS), left ventricular systolic inner diameter (LVIDs), and left ventricular diastolic inner diameter (LVIDd) values were recorded and compared to those of normal rats (all P > 0.05) (Fig. 4D).

To trace the expression of luciferase delivered by MN-AAV-LUC in living animals 4 weeks after MN-AAV administration, we applied a noninvasive small animal bioluminescence imaging system with high sensitivity. As shown in Fig. 4E (top), the AAV-LUC vectors transfected the target myocardium, resulting in high levels of luciferase expression in the heart, while no bioluminescence signals were detected in NC rats. In addition, proteins were extracted from the MN-AAV-LUC and NC groups, and an antifirefly luciferase antibody was used to detect the expression in Western blot (WB) assays (Fig. 4E, bottom), which indicated successful AAV delivery into and transfection of the myocardium.

To characterize the distribution of gene expression mediated by MN patches or DI, we analyzed rats that were subjected to gene delivery with AAV vectors encoding a GFP reporter gene. GFP-positive cells were detected in the anterior wall of the LV. The distribution of gene expression following the MN application was marked by an almost even distribution (Fig. 4F, top). In contrast, in the DI group, as described in previously published studies, the transfected cardiomyocytes were mainly confined to the site of the injection (Fig. 4F, middle). The distribution of fluorescent signals at five randomly selected horizontal lines in the fluorescent images was measured by ImageJ software (ImageJ 1.47v, National Institutes of Health). The results were plotted and fitted with OriginPro 8.5 software (OriginLab Corp., Northampton, MA, USA). The fluorescent signals were scattered evenly in the MN-AAV-GFP group, while in the DI-AAV-GFP group, the signals were confined to a specific region (Fig. 4F, bottom). The merged images of the two groups vividly demonstrated the variation in the distributions. No GFP-positive cells were found in the organs of lungs, kidneys, liver, or skeletal muscles, as shown in fig. S7. As indicated by representative in vivo images, no luciferase signals were observed in the defined organs, as shown in Fig. 4E (top).

To assess variations in cardiac function, we measured EF, FS, LVIDs, and LVIDd by echocardiography 2 days after left anterior descending coronary artery (LAD) ligation (baseline data) and 4 weeks after MN application (end point data). The study design for the AAV-VEGF treatment via MNs is illustrated in Fig. 5A. The parameters of the four groups (the MI, MI + MN, MI + DI-VEGF, and MI + MN-VEGF groups) measured at baseline did not differ significantly, indicating equivalent heart performance (fig. S8). Twenty-eight days after MN application, the rats with MI that received MN-VEGF had the greatest EF and FS values and the smallest LVIDs and LVIDd values (Fig. 5B and fig. S9A). EF was improved in the MN-AAV-VEGF group compared with the DI-AAV-VEGF (36.10 5.25% versus 30.29 2.10%, P = 0.042), MI (36.10 5.25% versus 24.28 4.34%, P = 0.0003), and MI + MN (36.10 5.25% versus 24.03 5.87%, P = 0.0002) groups. The MI + MN-VEGF group showed greater FS (18.28 2.97%) than the DI-AAV-VEGF group (15.04 1.05%, P = 0.0034), the MI + MN group (11.76 3.05%, P = 0.0002), and the MI group (11.93 2.27%, P = 0.0002). LVIDd and LVIDs in the MN-AAV-VEGF group were significantly lower than those in the MI group (LVIDd, 9.12 1.09 mm versus 10.55 0.69 mm, P = 0.0179; LVIDs, 7.59 1.01 mm versus 9.33 0.81 mm, P = 0.0048). The absolute changes in heart function (EF and FS) are shown in Fig. 5B. The MI and MI + MN groups showed significantly worse cardiac function in terms of the two parameters than the DI-AAV-VEGF and MI + MN-VEGF groups (EF, 18.77 2.36% in the MI group versus 6.07 4.63% in the MI + MN-VEGF group, P < 0.0001; FS, 9.97 1.25% in the MI group versus 3.14 2.48% in the MI + MN-VEGF group, P < 0.0001). Compared with the DI-AAV-VEGF, MI, and MI + MN groups, the MN-AAV-VEGF group showed a lack of significant change in cardiac function. Thus, MN-mediated VEGF expression improved cardiac function.

(A) Schematic illustrating the study design involving MN-AAV-VEGF application and improvement of injured heart function. (B) Representative echocardiographic images of the experimental groups 4 weeks following MN application. Left ventricular function parameters (EF, FS, LVIDs, and LVIDd) and absolute changes in heart function (EF and FS) were also measured and compared among the three groups. n = 6 animals per group. (C) Representative Massons trichromestained myocardial sections 4 weeks after MN-AAV-VEGF application. The scar areas and infarct sizes were quantified on the basis of Massons trichromestained images. Scale bars, 1 mm. (D) Identification of collagens via picrosirius red staining among the three groups. Scale bars, 1 mm. Representative polarized light images of the picrosirius redstained sections were subjected to polarized light microscopy. Scale bars, 100 m. Histograms showing the comparisons of collagen content and the type I/type III collagen ratios among the three groups. Right: Representative fluorescence micrographs identifying type I collagen (green) and type III collagen (red); the nuclei were stained with DAPI (blue). n = 3 animals per group. Scale bars, 500 m. All data are reported as the means SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

The infarction size and scar area were measured according to our previously described methods (26). Massons trichrome staining and magnified images revealed the morphology and fibrosis of heart tissues (Fig. 5C and fig. S9B). Compared to those in the MI and MI + MN groups (10.04 0.94 mm2 and 9.92 1.54 mm2, respectively), the scar areas (Fig. 5C, bottom) in the MI + MN-VEGF group (7.02 0.85 mm2) and the MI + DI-VEGF group (8.47 0.82 mm2) were effectively controlled by MN-VEGF application (fig. S9B). There were significant differences between the MI + MN-VEGF group and the control group (MI + MN-VEGF versus MI, P = 0.0004; MI + MN-VEGF versus MI + MN, P = 0.0006; MI + MN-VEGF versus MI + DI-VEGF, P = 0.049). In addition, the infarct size (Fig. 5C, bottom) was not different between the MI (71.27 8.37%) and MI + MN (68.86 3.25%) groups (P = 0.6187). The infarct size was reduced in the AAV-VEGFtreated groups (56.48 5.64% in the MI + DI-VEGF group and 46.17 10.68% in the MI + MN-VEGF group) (P < 0.0001 in the MI + MN-VEGF group compared with the MI group, P = 0.0002 in the MI + MN-VEGF group compared with the MI + MN group, and P = 0.00458 in the MI + MN-VEGF group compared with the MI + DI-VEGF group).

Picrosirius red staining combined with polarization microscopy was used to examine collagen fibers and to quantitatively determine their levels and types in scars in the four groups (Fig. 5D, left, and fig. S9C). Type I collagen was identified by yellow or red staining, and type III collagen was indicated by green staining under polarized light. The total collagen content in the infarcted region was similar among the groups, indicating no difference in collagen deposition (61.42 10.24% in the MI group, 58.97 11.83% in the MI + MN group, 60.16 3.86% in the MI + DI-VEGF group, and 58.97 11.83% in MI + MN-VEGF group, all P > 0.05). Moreover, the ratio of type I to type III collagen (type I/type III) was increased in the MI and MI + MN groups (3.63 3.79% versus 2.99 4.64%, P > 0.05). However, the ratio in the MI + MN-VEGF group (1.11 1.24%) was lower than those in the MI and MI + MN groups (P < 0.05). The ratio in the MI + DI-VEGF group (1.89 1.44%) was slightly higher than that in the MI + MN-VEGF group; however, this difference was not statistically significant (P = 0.2254). In addition, costaining of sections with type I (green) and type III (red) collagen antibodies was used for visualization of the collagen types (Fig. 5D, right, and fig. S9C).

To investigate the angiogenic and arteriogenic effects of MN-VEGF in the border zone and infarction region, we used antibodies against von Willebrand factor (vWF) and -smooth muscle actin (SMA) to stain endothelial cells and vascular smooth muscle cells, respectively. Tubular structures stained by fluorescent antibodies were identified as vessels. The capillary density was estimated on the basis of the vWF-positive vessels per HPF, and the arterial density was evaluated on the basis of SMA-positive vessels per HPF using the data collected at 4 weeks. The mature index was quantified as the ratio of SMA-positive vessels to the total number of vessels (27). As illustrated in Fig. 6B and fig. S9D, in the infarction region, the capillary density in the MI group was identical to that in the MI + MN group (8.33 1.51 per HPF versus 8.33 1.97 per HPF, P > 0.05). However, the value in the MI + MN-VEGF group (39.67 11.15 per HPF) significantly differed from that in the MI (P < 0.0001), MI + MN (P < 0.0001), and MI + DI-VEGF (25.83 5.19 per HPF, P = 0.0011) groups. Regarding the capillary density in the border zone, no difference was found between the MI and MI + MN groups (14.17 1.72 per HPF in the MI group and 14.17 2.40 per HPF in the MI + MN group, P > 0.05). The capillary density in the MI + MN-VEGF group was 72.67 13.46 per HPF (P < 0.0001 compared to those in the MI and MI + MN groups and P = 0.0002 compared to that in the MI + DI-VEGF group). The arterial density was compared as shown in Fig. 6B and fig. S9D. Compared to the MI + MN-VEGF group (38.83 9.77 per HPF, all P < 0.0001), the MI and MI + MN groups (3.83 1.72 per HPF in the MI group and 3.67 1.03 per HPF in the MI + MN group, P > 0.05) showed decreases of 90% in the infarction region. In addition, the arterial density of the MI + MN-VEGF group was significantly higher than that of the MI + DI-VEGF group (24.50 4.85 per HPF, P = 0.0012). In the border region, the arterial density was 6.67 2.50 per HPF in the MI group and 7.17 2.23 per HPF in the MI + MN group (P > 0.05). In the MI + MN-VEGF group, the arterial density was 66.83 12.86 per HPF (P < 0.0001 compared to those in the MI and MI + MN groups and P = 0.0025 compared to that the MI + DI-VEGF group). As shown in Fig. 6B and fig. S9D, the mature index in the infarction region was 36.55 11.60% in the MI group and 37.40 10.53% in the MI + MN group, with no difference between the two groups. In the MI + MN-VEGF group, the value was 86.3 1.67%, which was better than that in the MI (P < 0.0001), MI + MN (P < 0.0001), and MI + DI-VEGF (83.88 5.41%, P > 0.05) groups. No significant difference in the mature index was observed between the MI + MN-VEGF (85.20 4.46%) and MI + DI-VEGF (84.53 6.24%, P > 0.05) groups. The mature index in the MI + MN-VEGF group was markedly greater than that in the MI (39.97 13.85%) and MI + MN (41.73 9.23%) groups (all P < 0.0001). No significant differences in serum VEGF levels were detected at various time points between the MN-VEGF and MI groups (all P > 0.05) (Fig. 6C). The samples were measured in duplicate.

(A) Representative immunofluorescent images of vWF (green) and SMA (red) in the tissues of the infarction and border region showing increased vessel density in the MN-AAV-VEGF group compared with those in the other two groups. n = 3 animals per group. Scale bars, 50 m. Vessels are indicated by white triangles. (B) Quantification of capillary density, arterial density, and the mature index among the three groups in the infarction and border regions. (C) VEGF levels were detected by enzyme-linked immunosorbent assay (ELISA) in serum from the MI + MN-VEGF and MI groups. n = 3 animals per group. (D) Representative WB results for VEGF, VEGF receptor (VEGFR), phosphoinositide 3-kinase (PI3K), Akt, phosphorylated Akt (p-Akt), and caspase-9 in heart homogenates from the MI + MN-VEGF and MI groups. n = 3 animals per group. The bar graphs show the quantified protein levels. All data are reported as the means SD. *P < 0.05 and ****P < 0.0001.

The binding of VEGF to VEGF receptor 2 (VEGFR2) leads to the activation of diverse intracellular extracellular signaling pathways. WB analysis (Fig. 6D) showed that VEGF and VEGFR2, the high-affinity receptor of VEGF, were significantly up-regulated (all P < 0.05). The Akt and phosporylated Akt protein levels in AAV-treated hearts were significantly higher than those in non-AAVtreated MI hearts (all P < 0.05). The protein level of phosphoinositide 3-kinase (PI3K) in the MI + MN-VEGF group was increased, although the difference was not significant (P > 0.05). The level of the proapoptotic protein caspase 9 was significantly decreased in the MI + MN-VEGF group (P < 0.05).

CVD is the primary cause of mortality worldwide (28). Intramyocardial injection of therapeutic agents is a treatment strategy for patients suffering from this disease (29). Although local injection is a commonly used administration method to deliver agents to the myocardium, the effects are inevitably restricted to the injection site (911), which is attributed to the localized high concentrations of the agents. In addition, unlike in other organs, the injected agents can be extruded from the myocardium due to continuous dynamic muscle contraction. As reported in the published literature, a primary obstacle to cell therapy is the extremely low rate of myocardial retention after intramyocardial injection (13). It has been reported that almost 5 to 15% of intramyocardially injected cells are retained within the myocardium (30, 31); thus, only a fraction of injected cells contribute to therapeutic benefit. Development of new types of instruments and technologies to overcome this disadvantage is desperately needed. Figure S10 represents our vision for the clinical translation of MNs, in which MNs will be used to deliver therapeutic agents via a small thoracic incision to decrease the risk of infection induced by open-heart surgical procedures. Different from traditional approach of gene delivery, we developed an MN-AAV to deliver target gene into the myocardium. Coronary artery revascularization [percutaneous coronary intervention (PCI) or coronary-artery bypass grafting (CABG)] is an established therapeutic intervention. However, myocardial revascularization for ischemic regions with small coronary arteries remains a challenge in clinical practice. Gene therapy to improve vascular perfusion of those ischemic regions might be a promising alternative choice, especially for patients with IHD who are not candidates for PCI or CABG. In addition, because of the poor gene transfer efficiency in the myocardium and the inability of the therapy to target ischemic myocardium, the transduction efficiency was reduced. Thus, delivering MNs via less invasive surgeries repeatedly might improve the efficiency of gene transfection.

Angiogenic gene therapy for IHD is a promising option for the treatment of MI (32, 33). VEGF is important for the development and differentiation of the vascular network, with favorable preclinical evidence showing that it notably increases perfusion, improves tissue metabolism, improves cardiac function, and provides cardiac protection. However, intracoronary administration of VEGF protein has not yielded much clinical success (34). The principal limitation of administration of this protein is the short half-life of exogenous proteins in target tissue, which reduces the therapeutic benefit (35). To prolong the effects of angiogenic cytokines, recombinant plasmid DNA and viral vectors can be used, which allow for the consistent replication of the VEGF gene and maintain long-lasting protein expression in transfected cells. A series of studies have demonstrated improvement in rodent and large-animal (dog, sheep, and pig) models of ischemia and infarction following gene therapy with VEGF (3638). Similar results were obtained in our study. MN-AAV-VEGF ameliorated cardiac dysfunction in a rat model of MI. Therefore, the application of proangiogenic substances may be a new treatment option for patients with IHD. However, the results have not been very promising except for safety; follow-up conducted for over 10 years has indicated that there are no significant transgene or vector-related side effects (39).

On the other hand, previously published research has reported that high levels of circulating VEGF in acute MI can induce acute cor pulmonale, resulting in increased mortality (40). Unregulated and continuous expression of VEGF has been reported to lead to angioma formation at the site of injection (33, 41). However, in the future, cardiomyocyte-targeted viruses and improved gene transfection efficiency may enable the delivery of AAV vectors at low starting doses through repeated administration to control the expression of angiogenic factors. Furthermore, the side effects caused by VEGF overexpression might be ameliorated by regulation of gene expression, such as through gene switching, and other therapeutic approaches, such as antiangiogenic therapy using anti-VEGF antibodies. The management of angiogenic factor expression in both serum and target regions is important for enhancing the local therapeutic efficiency of this method and decreasing possible adverse effects (32). Consequently, careful application-specific consideration is warranted when selecting a processing strategy that minimizes unwanted responses.

To overcome difficult obstacles associated with the DI mode of agent administration, we fabricated MN-AAV to deliver the VEGF gene to rat hearts, which led to optimal distribution and local therapeutic efficiency. In addition, the coated vectors instantly penetrated into the myocardium, thus improving the retention of the delivered drugs, which indicated a better therapeutic effect in the MI + MN-VEGFtreated group than in the MI + DI-VEGF group. No significant differences in VEGF levels were detected at various time points between the MI + MN-VEGF and MI groups (Fig. 6C), similar to the results of experiments using large-animal models (32). In addition, no GFP or luciferase expression was detected in other organs (fig. S7 and Fig. 4E).

Versatile MN patches were fabricated according to our previously reported method (20) and were eventually machined into the desired sizes to achieve various characteristics, including sufficient strength to penetrate the target myocardium (Fig. 4B), water-swelling capacity (Fig. 2C), high drug-loading capacity, drug-loading uniformity (fig. S4), and therapeutic burst release kinetics (Fig. 2D). The phase transition capability allows efficient drug diffusion from a drug reservoir through a polymeric matrix with predictable accuracy (42). Researchers have long sought to control and overcome the burst release of agents during the application of MNs (43). However, this shortcoming was effectively used to deliver agents to the myocardium in this study. The intrinsic properties of the MNs and the modified AAV harboring approach resulted in a unique kinetic profile characterized by enhanced AAV delivery with predictable accuracy and early burst release kinetics. Extended release behavior in vitro was detected and confirmed by the AAV tilter assay (Fig. 2D). Combined with in vivo studies (Figs. 5 and 6), AAV-VEGFloaded MN can effectively ameliorate cardiac functions, reduce the scar size, and elevate myocardial perfusion in rat MI model, which suggested that MN-mediated gene delivery to targeted heart regions. Considering that we developed an MN-AAV to deliver gene vectors to repair injured myocardium and the isolated rat heart will suffer various pathophysiologic alterations after being removed from the living body, it is hard to simulate the complicated situations in the body by using Franz diffusion cells. Consequently, the release experiment by using Franz diffusion cell involved heart tissue is not conducted in our study. In addition, researchers have reported that the drug release results obtained using phosphate-buffered saline (PBS) and Franz diffusion cell were comparable (4446), indicating that these two experiments may be equivalent in representing the release profile of MNs. Regarding AAV loading, specific fluorescence imaging was absent in the control MNs, conversely, the surfaces of the` MN-FITC-AAV revealed a strong fluorescence signals (Fig. 2E), and the fluorescence intensity of MNs was identical among different patches, indicating the uniformity of drug loading in the MNs. In addition, the 3D images constructed by confocal microscopy confirmed that FITC-AAV was successfully and uniformly coated onto the surfaces of the MN bodies (Fig. 2F).

Hematoxylin and eosin (HE) staining of sections from hearts subjected to MN treatment revealed no signs of tissue necrosis, as shown in the representative sections. Although the wound area was relatively larger, the pinhole produced by each MN was quite small, and the tissue around each pinhole was not damaged (Fig. 4C). These results demonstrated that the wounds on the hearts might be acceptable and might self-heal after a period of time. Whether application in the hearts of large animals will result in any damage needs to be further studied. The inflammatory staining of the heart sections and the unaffected performance of the hearts treated with the MNs further confirmed the safe application of MNs (Fig. 4D and fig. S6). Previous research has suggested that MNs serve as channels connecting the patch and the host myocardium. For example, MN-loaded cardiac stem/stromal cells can secrete paracrine elements to treat injured hearts with good biocompatibility in rats (2). The spatial distribution of gene transfer mediated by MNs was also evaluated in this study. GFP-positive cells were detected and well distributed in the anterior wall of the LV after MN treatment (Fig. 4F, top). In contrast, in the DI group (Fig. 4F, middle), as described in previously published studies, the transfected cardiomyocytes were confined to the site of the injection (9, 10). MNs mediated gene delivery to the myocardium with a fine distribution and strong targeting precision. Analysis of MN-FITC-AAV and methylene bluestained MNs further confirmed the successful delivery of the released dyes and AAV particles into the myocardium with a homogeneous distribution (Fig. 4, B and C, and movies S3 and S4). The composite image of the in vivo imaging results also confirmed the targeted delivery of and transfection with the AAV-LUC vectors (Fig. 4E).

Given the safety and good distribution of MNs, we investigated the practicability of MN-mediated delivery of therapeutic agents, namely, AAV-VEGF, to the myocardium to treat injured hearts. First, the angiogenic effect of AAV-VEGF was tested in vitro. The HUVEC migration assay indicated that the culture medium of VEGF-transfected H9C2 cells had a powerful influence on the migration of HUVECs (Fig. 3D). The stimulation is an important step in neovessel formation (47). Then, MN-VEGF was used to treat the injured hearts. The echocardiographic results revealed significantly higher EF and FS values and significantly lower LVIDs and LVIDd values in the MN-VEGF group than in the DI-VEGF group and the other two control groups (Fig. 5B and fig. S9A), indicating functional improvement. No significant differences were observed in cardiac function between the MI and MI + MN groups. Significant decreases in the scar area and infarct size were observed in the MI + MN-VEGFtreated group, which showed outcomes superior to those in the three control groups (Fig. 5C). The increased expression of type I and type III collagen in infarcted zones has been suggested to protect hearts from remodeling and dilation (48). Although the differences among the four groups in total collagen content were not statistically significant, the ratio of type I to type III collagen was greater in the control groups than in the MI + MN-VEGFtreated group (Fig. 5D and fig. S9C), showing that the application of MN-VEGF modified the composition of collagen in the infarct scars (predominantly favoring type III collagen). Type III collagen confers elasticity and increases compliance (48), which might lead to the improvement of heart function.

Several studies have reported that VEGF expression improves cardiac function through the induction of angiogenesis (32, 49). Similar results were obtained in our study. As illustrated in Fig. 6A, compared with those in the control groups, the capillary and arterial densities in the scar tissue and border region were significantly increased in the MI + MN-VEGF group, which exhibited an elevated mature index. As previously reported, various signaling pathways, including the PI3K/Akt kinase pathway, were activated by the binding of VEGF to VEGFR, which can preserve cardiac performance (50). Consistent with the results from WB analysis of heart homogenates, the levels of many prosurvival proteins and a few proapoptotic proteins significantly differed in MI + MN-VEGFtreated hearts compared to MI hearts at 4 weeks after MN-VEGF application, indicating that signal transduction pathways were activated by the overexpression of VEGF (Fig. 6D).

This study has several limitations. First, the therapeutic effects of MN-VEGF were evaluated for 4 weeks in this study. In the future, longer time points should be used to determine the roles of MN-VEGF in regulating cardiac function. Second, to further broaden the clinical application of MNs, in vivo studies with large-animal models incorporating MN administration via minimally invasive surgery should be investigated.

In summary, we developed an AAV-loaded MN patch and showed that transepicardial permeation resulted in a homogeneous distribution of agents against direct local intramyocardial injection (after which the agents were confined to the site of injection). Our present study supports the practicality, safety, and versatility of MNs for delivery of therapeutic agents via minimally invasive surgery. This is a proof of concept study supporting translation to clinical applications.

The MNs were prepared according to our previously described method (20). These MNs are made of polyvinyl alcohol (PVA) and are not degradable. They will swell and dissolve after 6 months. The PVA can form microcrystalline domains as cross-linking junctions to produce a PTM patch. The PTM achieves highly efficient delivery of drugs and carriers without depositing the needle tip materials into the body. Briefly, patches were prepared with an air-permeable but water-impermeable mold. A vacuum was applied to the back to suck the aqueous PVA solutions into the holes within the mold. Then, a freeze-thaw process was conducted to form microcrystalline domains to enhance the mechanical strength of the MNs. Drying and punching processes were used after detaching the MNs from the molds (fig. S1). The mechanical properties of the MNs with or without AAV loading were assessed by a universal testing machine (MTS Echo, Exceed 40, USA) equipped with Test Suite TW software and a 100-N loading cell (51). In the compression assay, every patch was compressed to a strain of 20% at a rate of 10 mm/min with an initial load of 0.01 N. The compressive modulus was automatic calculated according to the GB/T 1041-2008/B/0 standard in the machine program. The differential strain (2 1) was 0.0025. A series of modulus was calculated at each point with strain of 0 to 20% and then was linearly fitted to obtain compressive modulus (n = 4 patches in each group). For testing and comparison of the swelling capability of the MNs, MN patches were immersed in PBS and incubated at 37C for at least 1 day until completely swelled; then, the MNs were photographed and measured. At least three measurements were taken, and mean values were reported. The fold change in the tip volume, which was based on the presumption of a conical shape, was calculated to reveal the phase-transition capability of MNs (52).

MN patches of the desired size (6 mm in diameter) were obtained with a punch. The patches were pierced through enlarged Parafilm (Parafilm M laboratory film) membranes (53). AAV9 vectors with cytomegalovirus (CMV) promoters containing the gene sequence for VEGF165 (AAV-VEGF) or GFP (AAV-GFP) alone were constructed by Shanghai GeneChem Co. Ltd. (Shanghai, China). An AAV9 vector with a CMV promoter containing the gene sequence for LUC (AAV-LUC) and an AAV2 vector with a CMV promoter containing the gene sequence for GFP (AAV2-GFP, which was used to assess the efficiency of transgene expression in vitro) were constructed by Hanbio (Shanghai, China). The AAV-containing solutions (~5 1010 vg) were dispensed to the patches and absorbed by the MN bodies. After completely drying in a customized low-temperature dryer, the film was peeled. The MN-AAV was used in in vitro and in vivo studies.

AAV was labeled with FITC (Thermo Fisher Scientific) according to a labeling protocol and a published paper (54). A Slide-A-Lyzer dialysis cassette (Slide-A-Lyzer Dialysis Cassette Kit, Invitrogen; 3.5K molecular weight cutoff, 3 ml) was applied to separate the unconjugated dyes. The yield of FITC conjugate was coated and immobilized onto the surfaces of the MNs with the aid of the intrinsic absorption capacity conferred by the phase-transition characterization.

MN patches were affixed to the inner caps of 1.5-ml Eppendorf microcentrifuge tubes filled with PBS. The tubes were inverted and incubated in a thermostatic shaker (37C with shaking at 100 rpm). The elution fluid was centrifuged to draw the solution from the cap to the base of the tube at 300g and collected at 2 s, 5 s, 10 s, 60 s, 100 s, 2 min, 6 min, and 1 day. Equal quantities of AAV were also detected as NCs. A scheme of these procedures is provided in fig. S2. In the published literature, real-time PCR has been applied to determine AAV titers (55). A real-time PCR assay of serial dilutions of plasmid vector standards and collected samples was performed in a Roche LC96 machine. The samples were preincubated at 95C for 3 min and then subjected to 40 cycles of 94C for 30 s (denaturation), 62C for 30 s (annealing), and 72C for 30 sec (amplification). The data were recorded as cycle threshold (Ct) values. Ct values are linearly correlated with the copy numbers of the templates in the exponential phase (55). The formula of the standard curve between the Ct value and the viral genome copy number was acquired from the standard samples. The titers of the released vectors in the samples were calculated according to the formula. The cumulative percentages of released vectors at different time intervals were calculated by dividing the values of the AAV quantity in the control group. The detection of each sample was performed in triplicate. In addition, the assay was repeated three independent experiments, and the mean values of each time point were used for graph plotting (Fig. 2D).

HEK 293 cells were cultured in Dulbeccos modified Eagles medium (Gibco, 11965092) supplemented with 10% fetal bovine serum (Gibco, 10270-106) and 1 penicillin-streptomycin (Gibco, 15070-063). Subconfluent 293 cells were seeded on the bottoms of the wells (Transwell culture inserts, pore size of 8 m; Corning, 3422). Virus-containing MNs (n = 3, each MN patch contained ~5 1010 vg AAV2-GFP) were placed on the filter inserts and incubated in medium for 1 hour (Fig. 3A). The percentages of 293 cells transfected by the released AAV2-GFP vectors were determined by flow cytometry of 10,000 cells (Beckman Coulter). In addition, mounted 293 cells grown on cleaned coverslips in cell culture dishes were imaged using fluorescence microscopy. We evaluated the transduction efficiency of MN-AAV before and after the freeze-thaw process. Subconfluent 293 cells were seeded on the bottoms of the wells. Virus-containing MNs (subjected and not subjected to the freeze-thaw process; n = 5 in each group) were placed on the filter inserts. After a 3-day incubation, the percentages of GFP-positive cells were determined by flow cytometry.

The culture supernatants of H9C2-VEGF cells transfected with released AAV-VEGF vectors and control groups (NC H9C2 cells and AAV-GFP transfected H9C2 cells) were collected. For migration assays, HUVECs (1 105 cells) in 200 l of culture medium without serum were added to Transwell inserts (Transwell culture inserts, pore size of 8 m; Corning, 3422), and 800 l of culture supernatant from each of the three groups was added to the lower Transwell chamber. The HUVECs were cultivated in a cell culture incubator for 20 hours. The cells were then fixed in methanol and stained with crystal violet solution (0.5%) at room temperature for 30 min. Cotton swabs were used to remove nonmigrated cells. The experiment was performed in triplicate. The transmigrated cells were photographed (200 magnification) with a Nikon Digital Sight DS-U2 (Nikon, Tokyo, Japan) camera attached to an Olympus BX50 microscope (Olympus Optical Co. Ltd., Tokyo, Japan). The total migrated HUVECs were quantified in five randomly selected HPFs. The HUVECs were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. The H9C2 cells were purchased from the American Type Culture Collection (CRL1446, cardiac myoblasts from rats).

Sprague-Dawley rats (male, 200 to 250 g) were obtained from the Shanghai Laboratory Animal Center. All procedures used in the study conformed to the Guide for the Care and Use of Laboratory Animals and were under the supervision of the Shanghai Jiao Tong University Institutional Animal Care and Use Committee. The Sprague-Dawley rats were anesthetized through intraperitoneal injection of pentobarbital sodium (30 mg/kg) and intubated with cannulas connected to a rodent ventilator. We previously applied MN patches to the skin by pressing with a thumb (at a force of 2.0 to 2.5 kg) (20). However, exertion of this heavy pressure to fix MNs on rat hearts while they are beating at 500 beats/min is difficult. Thus, a customized apparatus that operates via a principle similar to that of off-pump coronary aortic bypass grafting stabilizers (which provide stability during coronary revascularization surgery in patients suffering from CVD) was used for MN implantation (Fig. 1, A and C, and movies S1 and S3). The customized apparatus was a cylindrical conducting cavity with a backing plate. The inner and outer diameters were 9.2 and 10.3 mm, respectively. The inside cavity was 2.2 mm in height. An orifice (2.7 mm in diameter) was located in the center of the backing plate and was attached with a suction tube to a suction source. The MNs were attached to the backing plate with adhesive tape. There was a small gap (1.0 mm) between the hard backing of the MNs and the backing plate to ensure the patency of the cavity. When suction was provided, the targeted myocardial region entered the cavity of the customized apparatus. Consequently, MNs were passively and completely inserted into the soft myocardium. After stopping the supply of suction, the MNs detached from the backing plate and were maintained on the surface of the epicardium for 6 min. The negative pressure was ~8 kPa (~60 mmHg) at the time of MN application. The suction intensity (~400 mmHg) applied to immobilize beating hearts during coronary artery bypass surgery is clinically safe and does not cause myocardial damage (56). In the DI group, 50 l (~5 1010 vg) of AAV-GFP and AAV-VEGF, the same quantity of virus as that used in the MN-AAV group was injected into the left anterior wall in three equal aliquots using a 27-gauge needle via three injections into the predesignated area (57). The success of LAD ligation, which was used to induce the MI model, was confirmed by regional cyanosis of the anterior LV and an increase in the ST segment in the electrocardiogram (26). MN and MN-VEGF were implanted following MI. Echocardiographic measurements were taken for the four groups (the MI, MI + MN, MI + MN-VEGF, and MI + DI-VEGF groups) 2 days and 4 weeks after surgery. Isoflurane anesthesia was used to perform standard transthoracic echocardiography using an ultrasound imaging system (Vevo 2100 Imaging System, Visual Sonics, Toronto, ON, Canada). To assess cardiac function, echocardiographic data, including EF, FS, LVIDs, and LVIDd values, were collected and analyzed.

Methylene blue and FITC-AAVloaded MNs were used to confirm the insertion of the MNs following application to target heart regions in vivo. For analysis of MN insertion, the delivery of methylene blue and FITC-AAV to precise regions of the heart was assessed by observing and quantifying the puncture spots in the epicardium and heart sections. MN-AAV-LUC were applied to the left anterior wall in normal rats and detected by an in vivo fluorescence imaging system after 4 weeks. The luciferase activity in the region of interest was analyzed after intraperitoneal injection of the XenoLight d-Luciferin - K+ Salt bioluminescent substrate and detection with an in vivo bioluminescence imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA, USA) 10 min after the injection of substrate.

We compared the myocardial tissue density of CD68a pan-macrophage marker-positive macrophages among the MN-AAV, DI-AAV, and NC groups 7 days after the AAV vectors were delivered to the myocardium. In addition, cardiac function was measured in the rats that received the MNs, and normal rats were used as controls.

LV walls transfected with AAV-GFP and other rat organs, including the kidneys, lungs, liver, and skeletal muscles, were harvested at the end of the functional experiments, embedded, and frozen in Tissue-Tek optimum cutting temperature compound. Then, the walls were cryosectioned horizontally at an 8-m thickness, and 4% paraformaldehyde was applied to fix the heart samples for 3 days at 4C. The fixed tissues were embedded in paraffin and sectioned at a thickness of 4 m. The sections were placed onto slides and used for picrosirius red, HE, Massons trichrome, and immunofluorescence staining. WB assays were performed with standard WB techniques, as previously described (58), and the antigen-antibody reactions were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL, USA). The antibodies used in the current study are shown in table S1. Quantification was performed by densitometry. Independent experiments were repeated in triplicate. The tissue sections were stained with primary antibodies and then incubated with fluorescent secondary antibodies. The fluorescent images were acquired under a Zeiss LSM 700 confocal microscope or a fluorescence microscope.

For analysis of variations in serum VEGF, blood samples were collected from the MI and MI + MN-VEGF groups 0, 1, 2, 3, and 4 weeks after LAD ligation. The levels of serum VEGF were detected with an enzyme-linked immunosorbent assay (ELISA) kit for human VEGF (R&D Systems) according to a standard protocol and the manufacturers specifications. The samples were measured in duplicate.

Statistical analysis was performed using IBM SPSS software version 23.0. The data are presented as the means SD. The P values were calculated using one-way analysis of variance (ANOVA) with post hoc least significant difference multiple comparison tests to compare four groups or Students t test to compare two groups. The criterion of statistical significance was set at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: Funding: This work was supported by grants from the National Natural Science Foundation of China (81671832, 81571826, and 81690262), the Natural Science Foundation of Shanghai (18ZR1401900), the Shanghai Municipal Education CommissionGaofeng Clinical Medicine Grant Support (826158), and the Shanghai Municipal Key Clinical Specialty Construction Project. Author contributions: H.S. and T.X. contributed equally to this work. H.S., T.X., T.J., F.W., X.Y., and Q.Z. designed the research. H.S., T.X., C.J., S.H., Q.Y., and Y.Y. performed the cellular and animal experiments, analyzed the data, and drafted the paper. D.L. and Z.Y. performed the test of mechanical properties. Q.Z., X.Y., F.W., and T.J. directed and supervised the study. All authors contributed to the scientific discussions, data interpretation, and the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Microneedle-mediated gene delivery for the treatment of ischemic myocardial disease - Science Advances

Scientists Find This Relatively Harmless Virus Can Attack and Damage Human Heart – International Business Times, Singapore Edition

The world is increasingly becoming aware of the various kinds of damages that the SARS-CoV-2 can cause. However, researchers from Virginia Tech have found that the relatively harmless Adenovirus can cause heart conditions, which can be as life-threatening as the one induced by COVID-19.

According to the first-of-its-kind study, adenovirus can hamper the electrical signaling pathways between cells in the heart and also impair the ability of the cell to make new communication channels. The scientists exposed heart cells to the virus and learned of the potent effects it had on them.

"This is the first time we're putting this human virus on human heart cells to see what it does in the context of infected heart muscle cells. That's the real power of this," James Smyth, lead author of the study, said.

Adenoviruses belong to a class of common viruses that cause infections in the lining of the lungs, eyes, nervous system, and urinary tract. They often give rise to coughs, fever, pink eye, and sore throats, among others. While it generally affects children, all are prone to it.

The communication between heart muscles takes place through channels called gap junctions. They are formed by proteins known as connexins. Creating a bridge between two cells, gap junctions leads to the sharing of electrical signals that aid in the rhythmic contraction of the heart muscle cells. However, gap junctions can also alert neighboring cells about viral attacks.

Through the study, the researchers intended to demonstrate that the virus hijacks gap junctions, and when it does, it can decrease the production of connexin43(a component of a gap function). This in turn interrupts the electrical system that enables regular functioning of the heart, leading to arrhythmias (irregular heartbeat), and in extreme cases, cardiac death.

The researchers designed a diagnostic technique that employed pluripotent stem cell derived-cardiomyocytes, which are skin cells that have been made to convert to heart cells. The adenovirus was then applied to the cardiomyocytes and the resulting interactions were observed.

As expected, the virus hijacked the gap junctions in order to facilitate its own replication. However, the scientists also observed something that they had not anticipated. It was noted that two distinct processes were being carried out by the virus and that it inflicted dual damage to the cell's capacity to communicate with their neighbors. "Firstly, it was rapidly closing existing channels, and secondly it was shutting down the cells' ability to make new ones," explained Patrick Calhoun, co-author of the study.

Another aspect that caught the eye of the authors was the manner in which the virus prevented the creation of connexin43 and the formation of gap junctions. A protein pathway that is conventionally associated with the making of fresh connexin, was instead made to suppress its production by the virus. "We might learn something very new about the molecular biology there that's causing that switch," Smyth said

Smyth admits that the research is bound by the limitations of extending the results to a living heart while the experiment was conducted in vitro. However, highlighting the potential value of the findings, he asserted, "Fundamental studies provide the footing for the translational research that discovers therapeutics and diagnostic methods that improve people's health."

Going beyond the sheer understanding of viral infection, the research, Calhoun emphasized, can generate new therapeutic interventions for diseased hearts. "We're essentially learning from adenovirus to find the most efficient ways to stop, rather than cause, arrhythmias," he stressed.

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Scientists Find This Relatively Harmless Virus Can Attack and Damage Human Heart - International Business Times, Singapore Edition

Biomedicals big year: Grants fund research on skin, heart cells, cancer and more – Binghamton University

By Chris Kocher

June 18, 2020

The Thomas J. Watson School of Engineering and Applied Sciences Department of Biomedical Engineering has earned nearly $4 million in grants from 201820 (as of March 2020). Associate Professor Sha Jin alone received three grants totaling $1.2 million for her diabetes research. Funding agencies include the National Institutes of Health, the National Science Foundation and the National Institute of Standards and Technology.

Guy German

ASSOCIATE PROFESSOR

RESEARCH TOPIC: HUMAN SKIN

THE GOAL: Understanding how different factors can cause the mechanical properties of our skin to change. The human body has many barriers, and skin is arguably the most important, protecting us from the external environment. When skin becomes broken or ruptured, that barrier is lost. It can be caused by surgical incisions, penetrating trauma, diseases that cause lesions and chapping from cold environments. German explores how bacteria can degrade integrity; the effects of chronological- and photo-aging; and how to create bio-inspired materials that control crack propagation and the movement of fluids on their surfaces.

Tracy Hookway

ASSISTANT PROFESSOR

RESEARCH TOPIC: HEART CELLS

THE GOAL: Turning stem cells into functioning cardiac cells.

The human heart does not have the ability to repair itself after heart attacks or similar cardiac events. By merging the fields of stem-cell biology, tissue engineering and cardiovascular physiology, Hookway is trying to make models of cardiovascular tissue in a Petri dish that are more similar to what is in our bodies. One challenge is that the heart is not one cell type; in fact, it is multiple types of cells working together to achieve function.

Sha Jin

ASSOCIATE PROFESSOR

RESEARCH TOPIC: DIABETES

THE GOAL: Generating pancreatic tissue from stem cells.

One experimental treatment for diabetes currently in clinical trials through the U.S. Food and Drug Administration is islet transplantation, but there are fewer donors than needed. Human-induced pluripotent stem cells cells that can self-renew by dividing could offer a renewable source for islets, but they remain a challenge because of limited knowledge about how islets form. Jins lab has been working to direct stem cells to differentiate and mature into pancreatic islet organoids using a variety of approaches; when successful, these islets would be transplanted into humans.

Ahyeon Koh

ASSISTANT PROFESSOR

RESEARCH TOPIC: HUMAN SWEAT

THE GOAL: Utilizing sweat to generate electricity for flexible biosensors and to monitor stress levels.

Kohs research aims to give us real-time information about how our bodies are functioning, such as for glucose monitoring, wound care and post-surgery cardiac health. She is currently working with other Binghamton professors on two microfluidic systems that can collect and use the sweat that our body produces. One of them will have sweat-eating bacteria that will power biosensors, and the other will monitor stress levels by measuring the amounts of the steroid hormone cortisol that are secreted.

Gretchen Mahler

ASSOCIATE PROFESSOR

RESEARCH TOPIC: ORGAN-ON-A-CHIP

THE GOAL: Creating 3D microfluidic cell-culture chips that simulate the mechanics and physiological response of organs and tissues.

Mahlers current research which has applications for cardiovascular disease and cancer focuses on how disruptions in a tissues mechanical or chemical environment can lead to disease initiation and progression. She currently is working with three other professors two from Watson, one from Harpur College of Arts and Sciences on a National Science Foundation-funded study of calcific aortic valve disease, and she also is interested in how food additives alter gastrointestinal health.

Kaiming Ye

PROFESSOR AND DEPARTMENT CHAIR

RESEARCH TOPIC: CANCER VACCINE

THE GOAL: Developing a vaccine that will slow or halt the growth of future tumors.Yes research is targeting the protein CD47, which is part of the membrane that covers human cells. It also sends a dont eat me signal to a bodys immune system normally a good thing, but a problem when cells become cancerous. In a 2019 study using mice treated with their experimental vaccine, Ye and his co-investigators found a two-fold reduction in tumor growth rates and five-fold reduction in size in the tumors that did form.

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Biomedicals big year: Grants fund research on skin, heart cells, cancer and more - Binghamton University

Global Autologous Stem Cell Based Therapies Market 2020 Growth, Industry Trends, Sales Revenue, Size by Regional Forecast to 2025 – 3rd Watch News

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FDA Approves Second Biomarker-Based Indication for Merck’s KEYTRUDA (pembrolizumab), Regardless of Tumor Type – BioSpace

KENILWORTH, N.J.--(BUSINESS WIRE)-- Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the U.S. Food and Drug Administration (FDA) has approved KEYTRUDA, Mercks anti-PD-1 therapy, as monotherapy for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase (mut/Mb)] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Immune-mediated adverse reactions, which may be severe or fatal, can occur with KEYTRUDA, including pneumonitis, colitis, hepatitis, endocrinopathies, nephritis and renal dysfunction, severe skin reactions, solid organ transplant rejection, and complications of allogeneic hematopoietic stem cell transplantation (HSCT). Based on the severity of the adverse reaction, KEYTRUDA should be withheld or discontinued and corticosteroids administered if appropriate. KEYTRUDA can also cause severe or life-threatening infusion-related reactions. Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. For more information, see Selected Important Safety Information below.

For the second time, KEYTRUDA monotherapy is now approved based on a biomarker rather than the location in the body where the tumor originated, said Dr. Scot Ebbinghaus, vice president, clinical research, Merck Research Laboratories. TMB-H, defined as 10 mutations per megabase or more, can help identify patients most likely to benefit from KEYTRUDA. Were pleased that our collaborative efforts to advance biomarker research have resulted in our ability to provide a new treatment option that addresses a high unmet medical need for these patients with cancer.

As physicians, we are always looking to find new options for patients, especially in the second-line or higher treatment setting, said Roy S. Herbst, M.D., Ph.D., ensign professor of medicine (medical oncology) and professor of pharmacology, Yale School of Medicine; chief of medical oncology, Yale Cancer Center and Smilow Cancer Hospital; and associate cancer center director for translational research, Yale Cancer Center. Its great to see the use of innovative biomarkers and immunotherapy come together with this approval and encouraging that we now have an option for patients with TMB-H tumors across cancer types, including rare cancers.

The FDA also approved FoundationOne CDx test as the companion diagnostic to identify patients with solid tumors that are TMB-H (10 mutations/ megabase) who may benefit from immunotherapy treatment with KEYTRUDA monotherapy.

These approvals stem from years of research into how TMB levels may influence a patients response to immunotherapy, said Brian Alexander, M.D., M.P.H., chief medical officer, Foundation Medicine. Its critical that healthcare professionals have access to a validated genomic test to measure TMB in clinical tumor assessments and pinpoint those who are more likely to respond. Were proud to be collaborating with Merck to help match appropriate patients to this important treatment.

Data Supporting the Approval

The accelerated approval was based on data from a prospectively-planned retrospective analysis of 10 cohorts (A through J) of patients with various previously treated unresectable or metastatic solid tumors with TMB-H, who were enrolled in KEYNOTE-158 (NCT02628067), a multicenter, non-randomized, open-label trial evaluating KEYTRUDA (200 mg every three weeks). The trial excluded patients who previously received an anti-PD-1 or other immune-modulating monoclonal antibody, or who had an autoimmune disease, or a medical condition that required immunosuppression. TMB status was assessed using the FoundationOne CDx assay and pre-specified cutpoints of 10 and 13 mut/Mb, and testing was blinded with respect to clinical outcomes. Tumor response was assessed every nine weeks for the first 12 months and every 12 weeks thereafter. The major efficacy outcome measures were objective response rate (ORR) and duration of response (DOR) in the patients who received at least one dose of KEYTRUDA as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, modified to follow a maximum of 10 target lesions and a maximum of five target lesions per organ.

In KEYNOTE-158, 1,050 patients were included in the efficacy analysis population. TMB was analyzed in the subset of 790 patients with sufficient tissue for testing based on protocol-specified testing requirements. Of the 790 patients, 102 (13%) had tumors identified as TMB-H, defined as TMB 10 mut/Mb. The study population characteristics of these 102 patients were: median age of 61 years (range, 27 to 80); 34% age 65 or older; 34% male; 81% White; and 41% Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) of 0 and 58% ECOG PS of 1. Fifty-six percent of patients had at least two prior lines of therapy.

In the 102 patients whose tumors were TMB-H, KEYTRUDA demonstrated an ORR of 29% (95% CI, 21-39), with a complete response rate of 4% and a partial response rate of 25%. After a median follow-up time of 11.1 months, the median DOR had not been reached (range, 2.2+ to 34.8+ months). Among the 30 responding patients, 57% had ongoing responses of 12 months or longer, and 50% had ongoing responses of 24 months or longer.

In a pre-specified analysis of patients with TMB 13 mut/Mb (n=70), KEYTRUDA demonstrated an ORR of 37% (95% CI, 26-50), with a complete response rate of 3% and a partial response rate of 34%. After a median follow-up time of 11.1 months, the median DOR had not been reached (range, 2.2+ to 34.8+ months). Among the 26 responding patients, 58% had ongoing responses of 12 months or longer, and 50% had ongoing responses of 24 months or longer. In an exploratory analysis in 32 patients whose cancer had TMB 10 mut/Mb and <13 mut/Mb, the ORR was 13% (95% CI, 4-29), including two complete responses and two partial responses.

The median duration of exposure to KEYTRUDA was 4.9 months (range, 0.03 to 35.2 months). The most common adverse reactions for KEYTRUDA (reported in 20% of patients) were fatigue, musculoskeletal pain, decreased appetite, pruritus, diarrhea, nausea, rash, pyrexia, cough, dyspnea, constipation, pain and abdominal pain.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,200 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory classical Hodgkin lymphoma (cHL), or who have relapsed after 3 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 [combined positive score (CPS) 10], as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High (MSI-H) Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase (mut/Mb)] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options.

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Selected Important Safety Information for KEYTRUDA

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis, including fatal cases. Pneumonitis occurred in 3.4% (94/2799) of patients with various cancers receiving KEYTRUDA, including Grade 1 (0.8%), 2 (1.3%), 3 (0.9%), 4 (0.3%), and 5 (0.1%). Pneumonitis occurred in 8.2% (65/790) of NSCLC patients receiving KEYTRUDA as a single agent, including Grades 3-4 in 3.2% of patients, and occurred more frequently in patients with a history of prior thoracic radiation (17%) compared to those without (7.7%). Pneumonitis occurred in 6% (18/300) of HNSCC patients receiving KEYTRUDA as a single agent, including Grades 3-5 in 1.6% of patients, and occurred in 5.4% (15/276) of patients receiving KEYTRUDA in combination with platinum and FU as first-line therapy for advanced disease, including Grades 3-5 in 1.5% of patients.

Monitor patients for signs and symptoms of pneumonitis. Evaluate suspected pneumonitis with radiographic imaging. Administer corticosteroids for Grade 2 or greater pneumonitis. Withhold KEYTRUDA for Grade 2; permanently discontinue KEYTRUDA for Grade 3 or 4 or recurrent Grade 2 pneumonitis.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis. Colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 2 (0.4%), 3 (1.1%), and 4 (<0.1%). Monitor patients for signs and symptoms of colitis. Administer corticosteroids for Grade 2 or greater colitis. Withhold KEYTRUDA for Grade 2 or 3; permanently discontinue KEYTRUDA for Grade 4 colitis.

Immune-Mediated Hepatitis (KEYTRUDA) and Hepatotoxicity (KEYTRUDA in Combination With Axitinib)

Immune-Mediated Hepatitis

KEYTRUDA can cause immune-mediated hepatitis. Hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.4%), and 4 (<0.1%). Monitor patients for changes in liver function. Administer corticosteroids for Grade 2 or greater hepatitis and, based on severity of liver enzyme elevations, withhold or discontinue KEYTRUDA.

Hepatotoxicity in Combination With Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity with higher than expected frequencies of Grades 3 and 4 ALT and AST elevations compared to KEYTRUDA alone. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased ALT (20%) and increased AST (13%) were seen. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider more frequent monitoring of liver enzymes as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed.

Immune-Mediated Endocrinopathies

KEYTRUDA can cause adrenal insufficiency (primary and secondary), hypophysitis, thyroid disorders, and type 1 diabetes mellitus. Adrenal insufficiency occurred in 0.8% (22/2799) of patients, including Grade 2 (0.3%), 3 (0.3%), and 4 (<0.1%). Hypophysitis occurred in 0.6% (17/2799) of patients, including Grade 2 (0.2%), 3 (0.3%), and 4 (<0.1%). Hypothyroidism occurred in 8.5% (237/2799) of patients, including Grade 2 (6.2%) and 3 (0.1%). The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC (16%) receiving KEYTRUDA, as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. Hyperthyroidism occurred in 3.4% (96/2799) of patients, including Grade 2 (0.8%) and 3 (0.1%), and thyroiditis occurred in 0.6% (16/2799) of patients, including Grade 2 (0.3%). Type 1 diabetes mellitus, including diabetic ketoacidosis, occurred in 0.2% (6/2799) of patients.

Monitor patients for signs and symptoms of adrenal insufficiency, hypophysitis (including hypopituitarism), thyroid function (prior to and periodically during treatment), and hyperglycemia. For adrenal insufficiency or hypophysitis, administer corticosteroids and hormone replacement as clinically indicated. Withhold KEYTRUDA for Grade 2 adrenal insufficiency or hypophysitis and withhold or discontinue KEYTRUDA for Grade 3 or Grade 4 adrenal insufficiency or hypophysitis. Administer hormone replacement for hypothyroidism and manage hyperthyroidism with thionamides and beta-blockers as appropriate. Withhold or discontinue KEYTRUDA for Grade 3 or 4 hyperthyroidism. Administer insulin for type 1 diabetes, and withhold KEYTRUDA and administer antihyperglycemics in patients with severe hyperglycemia.

Immune-Mediated Nephritis and Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.1%), and 4 (<0.1%) nephritis. Nephritis occurred in 1.7% (7/405) of patients receiving KEYTRUDA in combination with pemetrexed and platinum chemotherapy. Monitor patients for changes in renal function. Administer corticosteroids for Grade 2 or greater nephritis. Withhold KEYTRUDA for Grade 2; permanently discontinue for Grade 3 or 4 nephritis.

Immune-Mediated Skin Reactions

Immune-mediated rashes, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) (some cases with fatal outcome), exfoliative dermatitis, and bullous pemphigoid, can occur. Monitor patients for suspected severe skin reactions and based on the severity of the adverse reaction, withhold or permanently discontinue KEYTRUDA and administer corticosteroids. For signs or symptoms of SJS or TEN, withhold KEYTRUDA and refer the patient for specialized care for assessment and treatment. If SJS or TEN is confirmed, permanently discontinue KEYTRUDA.

Other Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue in patients receiving KEYTRUDA and may also occur after discontinuation of treatment. For suspected immune-mediated adverse reactions, ensure adequate evaluation to confirm etiology or exclude other causes. Based on the severity of the adverse reaction, withhold KEYTRUDA and administer corticosteroids. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Based on limited data from clinical studies in patients whose immune-related adverse reactions could not be controlled with corticosteroid use, administration of other systemic immunosuppressants can be considered. Resume KEYTRUDA when the adverse reaction remains at Grade 1 or less following corticosteroid taper. Permanently discontinue KEYTRUDA for any Grade 3 immune-mediated adverse reaction that recurs and for any life-threatening immune-mediated adverse reaction.

The following clinically significant immune-mediated adverse reactions occurred in less than 1% (unless otherwise indicated) of 2799 patients: arthritis (1.5%), uveitis, myositis, Guillain-Barr syndrome, myasthenia gravis, vasculitis, pancreatitis, hemolytic anemia, sarcoidosis, and encephalitis. In addition, myelitis and myocarditis were reported in other clinical trials, including classical Hodgkin lymphoma, and postmarketing use.

Treatment with KEYTRUDA may increase the risk of rejection in solid organ transplant recipients. Consider the benefit of treatment vs the risk of possible organ rejection in these patients.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% (6/2799) of patients. Monitor patients for signs and symptoms of infusion-related reactions. For Grade 3 or 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Immune-mediated complications, including fatal events, occurred in patients who underwent allogeneic HSCT after treatment with KEYTRUDA. Of 23 patients with cHL who proceeded to allogeneic HSCT after KEYTRUDA, 6 (26%) developed graft-versus-host disease (GVHD) (1 fatal case) and 2 (9%) developed severe hepatic veno-occlusive disease (VOD) after reduced-intensity conditioning (1 fatal case). Cases of fatal hyperacute GVHD after allogeneic HSCT have also been reported in patients with lymphoma who received a PD-1 receptorblocking antibody before transplantation. Follow patients closely for early evidence of transplant-related complications such as hyperacute graft-versus-host disease (GVHD), Grade 3 to 4 acute GVHD, steroid-requiring febrile syndrome, hepatic veno-occlusive disease (VOD), and other immune-mediated adverse reactions.

In patients with a history of allogeneic HSCT, acute GVHD (including fatal GVHD) has been reported after treatment with KEYTRUDA. Patients who experienced GVHD after their transplant procedure may be at increased risk for GVHD after KEYTRUDA. Consider the benefit of KEYTRUDA vs the risk of GVHD in these patients.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with a PD-1 or PD-L1 blocking antibody in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-002, KEYTRUDA was permanently discontinued due to adverse reactions in 12% of 357 patients with advanced melanoma; the most common (1%) were general physical health deterioration (1%), asthenia (1%), dyspnea (1%), pneumonitis (1%), and generalized edema (1%). The most common adverse reactions were fatigue (43%), pruritus (28%), rash (24%), constipation (22%), nausea (22%), diarrhea (20%), and decreased appetite (20%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

In KEYNOTE-189, when KEYTRUDA was administered with pemetrexed and platinum chemotherapy in metastatic nonsquamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 20% of 405 patients. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonitis (3%) and acute kidney injury (2%). The most common adverse reactions (20%) with KEYTRUDA were nausea (56%), fatigue (56%), constipation (35%), diarrhea (31%), decreased appetite (28%), rash (25%), vomiting (24%), cough (21%), dyspnea (21%), and pyrexia (20%).

In KEYNOTE-407, when KEYTRUDA was administered with carboplatin and either paclitaxel or paclitaxel protein-bound in metastatic squamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 15% of 101 patients. The most frequent serious adverse reactions reported in at least 2% of patients were febrile neutropenia, pneumonia, and urinary tract infection. Adverse reactions observed in KEYNOTE-407 were similar to those observed in KEYNOTE-189 with the exception that increased incidences of alopecia (47% vs 36%) and peripheral neuropathy (31% vs 25%) were observed in the KEYTRUDA and chemotherapy arm compared to the placebo and chemotherapy arm in KEYNOTE-407.

In KEYNOTE-042, KEYTRUDA was discontinued due to adverse reactions in 19% of 636 patients with advanced NSCLC; the most common were pneumonitis (3%), death due to unknown cause (1.6%), and pneumonia (1.4%). The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia (7%), pneumonitis (3.9%), pulmonary embolism (2.4%), and pleural effusion (2.2%). The most common adverse reaction (20%) was fatigue (25%).

In KEYNOTE-010, KEYTRUDA monotherapy was discontinued due to adverse reactions in 8% of 682 patients with metastatic NSCLC; the most common was pneumonitis (1.8%). The most common adverse reactions (20%) were decreased appetite (25%), fatigue (25%), dyspnea (23%), and nausea (20%).

Adverse reactions occurring in patients with SCLC were similar to those occurring in patients with other solid tumors who received KEYTRUDA as a single agent.

In KEYNOTE-048, KEYTRUDA monotherapy was discontinued due to adverse events in 12% of 300 patients with HNSCC; the most common adverse reactions leading to permanent discontinuation were sepsis (1.7%) and pneumonia (1.3%). The most common adverse reactions (20%) were fatigue (33%), constipation (20%), and rash (20%).

In KEYNOTE-048, when KEYTRUDA was administered in combination with platinum (cisplatin or carboplatin) and FU chemotherapy, KEYTRUDA was discontinued due to adverse reactions in 16% of 276 patients with HNSCC. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonia (2.5%), pneumonitis (1.8%), and septic shock (1.4%). The most common adverse reactions (20%) were nausea (51%), fatigue (49%), constipation (37%), vomiting (32%), mucosal inflammation (31%), diarrhea (29%), decreased appetite (29%), stomatitis (26%), and cough (22%).

In KEYNOTE-012, KEYTRUDA was discontinued due to adverse reactions in 17% of 192 patients with HNSCC. Serious adverse reactions occurred in 45% of patients. The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia, dyspnea, confusional state, vomiting, pleural effusion, and respiratory failure. The most common adverse reactions (20%) were fatigue, decreased appetite, and dyspnea. Adverse reactions occurring in patients with HNSCC were generally similar to those occurring in patients with melanoma or NSCLC who received KEYTRUDA as a monotherapy, with the exception of increased incidences of facial edema and new or worsening hypothyroidism.

In KEYNOTE-087, KEYTRUDA was discontinued due to adverse reactions in 5% of 210 patients with cHL. Serious adverse reactions occurred in 16% of patients; those 1% included pneumonia, pneumonitis, pyrexia, dyspnea, GVHD, and herpes zoster. Two patients died from causes other than disease progression; 1 from GVHD after subsequent allogeneic HSCT and 1 from septic shock. The most common adverse reactions (20%) were fatigue (26%), pyrexia (24%), cough (24%), musculoskeletal pain (21%), diarrhea (20%), and rash (20%).

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FDA Approves Second Biomarker-Based Indication for Merck's KEYTRUDA (pembrolizumab), Regardless of Tumor Type - BioSpace

Exosome Therapeutic Market 2020 Global Industry Growth, Size, Demand, Trends, Insights | Leading Players evox THERAPEUTICS, EXOCOBIO, Exopharm, AEGLE…

The Exosome Therapeutic Market study analyzes the market status, market share, growth rate, future trends, market drivers, opportunities, challenges, risks, entry barriers, sales channels, distributors & Porters Five Forces Analysis. This market report performs geographical analysis for the major areas such as North America, China, Europe, Southeast Asia, Japan, and India, with respect to the production, price, revenue and market share for top manufacturers. Moreover, businesses can gain insights into profit growth and sustainability program with this report. The Exosome Therapeutic Market report also consists detailed profiles of markets major manufacturers and importers who are dominating the market.

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Exosome therapeutic market is expected to gain market growth in the forecast period of 2019 to 2026. Data Bridge Market Research analyses that the market is growing with a CAGR of 21.9% in the forecast period of 2019 to 2026 and expected to reach USD 31,691.52 million by 2026 from USD 6,500.00 million in 2018. Increasing prevalence of lyme disease, chronic inflammation, autoimmune disease and other chronic degenerative diseases are the factors for the market growth.

The major players covered in the Exosome Therapeutic Market report are evox THERAPEUTICS, EXOCOBIO, Exopharm, AEGLE Therapeutics, United Therapeutics Corporation, Codiak BioSciences, Jazz Pharmaceuticals, Inc., Boehringer Ingelheim International GmbH, ReNeuron Group plc, Capricor Therapeutics, Avalon Globocare Corp., CREATIVE MEDICAL TECHNOLOGY HOLDINGS INC., Stem Cells Group among other players domestic and global. Exosome therapeutic market share data is available for Global, North America, Europe, Asia-Pacific, and Latin America separately. DBMR analysts understand competitive strengths and provide competitive analysis for each competitor separately.

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Exosomes are used to transfer RNA, DNA, and proteins to other cells in the body by making alteration in the function of the target cells. Increasing research activities in exosome therapeutic is augmenting the market growth as demand for exosome therapeutic has increased among healthcare professionals.

Increased number of exosome therapeutics as compared to the past few years will accelerate the market growth. Companies are receiving funding for exosome therapeutic research and clinical trials. For instance, In September 2018, EXOCOBIO has raised USD 27 million in its series B funding. The company has raised USD 46 million as series a funding in April 2017. The series B funding will help the company to set up GMP-compliant exosome industrial facilities to enhance production of exosomes to commercialize in cosmetics and pharmaceutical industry.

Increasing demand for anti-aging therapies will also drive the market. Unmet medical needs such as very few therapeutic are approved by the regulatory authority for the treatment in comparison to the demand in global exosome therapeutics market will hamper the market growth market. Availability of various exosome isolation and purification techniques is further creates new opportunities for exosome therapeutics as they will help company in isolation and purification of exosomes from dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, and urine and from others sources. Such policies support exosome therapeutic market growth in the forecast period to 2019-2026.

This exosome therapeutic market report provides details of market share, new developments, and product pipeline analysis, impact of domestic and localised market players, analyses opportunities in terms of emerging revenue pockets, changes in market regulations, product approvals, strategic decisions, product launches, geographic expansions, and technological innovations in the market. To understand the analysis and the market scenario contact us for anAnalyst Brief, our team will help you create a revenue impact solution to achieve your desired goal.

Global Exosome Therapeutic Market Scope and Market Size

Global exosome therapeutic market is segmented of the basis of type, source, therapy, transporting capacity, application, route of administration and end user. The growth among segments helps you analyse niche pockets of growth and strategies to approach the market and determine your core application areas and the difference in your target markets.

Based on type, the market is segmented into natural exosomes and hybrid exosomes. Natural exosomes are dominating in the market because natural exosomes are used in various biological and pathological processes as well as natural exosomes has many advantages such as good biocompatibility and reduced clearance rate compare than hybrid exosomes.

Exosome is an extracellular vesicle which is released from cells, particularly from stem cells. Exosome functions as vehicle for particular proteins and genetic information and other cells. Exosome plays a vital role in the rejuvenation and communication of all the cells in our body while not themselves being cells at all. Research has projected that communication between cells is significant in maintenance of healthy cellular terrain. Chronic disease, age, genetic disorders and environmental factors can affect stem cells communication with other cells and can lead to distribution in the healing process. The growth of the global exosome therapeutic market reflects global and country-wide increase in prevalence of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases, along with increasing demand for anti-aging therapies. Additionally major factors expected to contribute in growth of the global exosome therapeutic market in future are emerging therapeutic value of exosome, availability of various exosome isolation and purification techniques, technological advancements in exosome and rising healthcare infrastructure.

Rising demand of exosome therapeutic across the globe as exosome therapeutic is expected to be one of the most prominent therapies for autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases treatment, according to clinical researches exosomes help to processes regulation within the body during treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases. This factor has increased the research activities in exosome therapeutic development around the world for exosome therapeutic. Hence, this factor is leading the clinician and researches to shift towards exosome therapeutic. In the current scenario the exosome therapeutic are highly used in treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases and as anti-aging therapy as it Exosomes has proliferation of fibroblast cells which is significant in maintenance of skin elasticity and strength.

Based on source, the market is segmented into dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, urine and others. Mesenchymal stem cells are dominating in the market because mesenchymal stem cells (MSCs) are self-renewable, multipotent, easily manageable and customarily stretchy in vitro with exceptional genomic stability. Mesenchymal stem cells have a high capacity for genetic manipulation in vitro and also have good potential to produce. It is widely used in treatment of inflammatory and degenerative disease offspring cells encompassing the transgene after transplantation.

Based on therapy, the market is segmented into immunotherapy, gene therapy and chemotherapy. Chemotherapy is dominating in the market because chemotherapy is basically used in treatment of cancer which is major public health issues. The multidrug resistance (MDR) proteins and various tumors associated exosomes such as miRNA and IncRNA are include in in chemotherapy associated resistance.

Based on transporting capacity, the market is segmented into bio macromolecules and small molecules. Bio macromolecules are dominating in the market because bio macromolecules transmit particular biomolecular information and are basically investigated for their delicate properties such as biomarker source and delivery system.

Based on application, the market is segmented into oncology, neurology, metabolic disorders, cardiac disorders, blood disorders, inflammatory disorders, gynecology disorders, organ transplantation and others. Oncology segment is dominating in the market due to rising incidence of various cancers such as lung cancer, breast cancer, leukemia, skin cancer, lymphoma. As per the National Cancer Institute, in 2018 around 1,735,350 new cases of cancer was diagnosed in the U.S. As per the American Cancer Society Inc in 2019 approximately 268,600 new cases of breast cancer diagnosed in the U.S.

Based on route of administration, the market is segmented into oral and parenteral. Parenteral route is dominating in the market because it provides low drug concentration, free from first fast metabolism, low toxicity as compared to oral route as well as it is suitable in unconscious patients, complicated to swallow drug etc.

The exosome therapeutic market, by end user, is segmented into hospitals, diagnostic centers and research & academic institutes. Hospitals are dominating in the market because hospitals provide better treatment facilities and skilled staff as well as treatment available at affordable cost in government hospitals.

Exosome therapeutic Market Country Level Analysis

The global exosome therapeutic market is analysed and market size information is provided by country by type, source, therapy, transporting capacity, application, route of administration and end user as referenced above.

The countries covered in the exosome therapeutic market report are U.S. and Mexico in North America, Turkey in Europe, South Korea, Australia, Hong Kong in the Asia-Pacific, Argentina, Colombia, Peru, Chile, Ecuador, Venezuela, Panama, Dominican Republic, El Salvador, Paraguay, Costa Rica, Puerto Rico, Nicaragua, Uruguay as part of Latin America.

Country Level Analysis, By Type

North America dominates the exosome therapeutic market as the U.S. is leader in exosome therapeutic manufacturing as well as research activities required for exosome therapeutics. At present time Stem Cells Group holding shares around 60.00%. In addition global exosomes therapeutics manufacturers like EXOCOBIO, evox THERAPEUTICS and others are intensifying their efforts in China. The Europe region is expected to grow with the highest growth rate in the forecast period of 2019 to 2026 because of increasing research activities in exosome therapeutic by population.

The country section of the report also provides individual market impacting factors and changes in regulation in the market domestically that impacts the current and future trends of the market. Data points such as new sales, replacement sales, country demographics, regulatory acts and import-export tariffs are some of the major pointers used to forecast the market scenario for individual countries. Also, presence and availability of global brands and their challenges faced due to large or scarce competition from local and domestic brands, impact of sales channels are considered while providing forecast analysis of the country data.

Huge Investment by Automakers for Exosome Therapeutics and New Technology Penetration

Global exosome therapeutic market also provides you with detailed market analysis for every country growth in pharma industry with exosome therapeutic sales, impact of technological development in exosome therapeutic and changes in regulatory scenarios with their support for the exosome therapeutic market. The data is available for historic period 2010 to 2017.

Competitive Landscape and Exosome Therapeutic Market Share Analysis

Global exosome therapeutic market competitive landscape provides details by competitor. Details included are company overview, company financials, revenue generated, market potential, investment in research and development, new market initiatives, global presence, production sites and facilities, company strengths and weaknesses, product launch, product trials pipelines, concept cars, product approvals, patents, product width and breadth, application dominance, technology lifeline curve. The above data points provided are only related to the companys focus related to global exosome therapeutic market.

Many joint ventures and developments are also initiated by the companies worldwide which are also accelerating the global exosome therapeutic market.

For instance,

Partnership, joint ventures and other strategies enhances the company market share with increased coverage and presence. It also provides the benefit for organisation to improve their offering for exosome therapeutics through expanded model range.

Customization Available:Global Exosome Therapeutic Market

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Exosome Therapeutic Market 2020 Global Industry Growth, Size, Demand, Trends, Insights | Leading Players evox THERAPEUTICS, EXOCOBIO, Exopharm, AEGLE...

Stem Cell Therapy Market Grows on Back of Growing Awareness Regarding Regenerative Treatment Methods – BioSpace

Lately, there has been rising awareness among people regarding the therapeutic potential of stem cells for disease management. This is one of the key factors contributing to growth of the global stem cell therapy market.

Further, identification of new stem cell lines, research and development of genome based cell analysis techniques, and investment inflow for processing and banking of stem cell are some of the significant factors augmenting expansion rate of the global stem cell therapy market.

Meanwhile, limitations associated with traditional organ transplantation such as immunosuppression risk, infection risk, and low acceptance rate of organ by body are few features leading to adoption of stem cell therapy. Moreover, high dependency on organ donors for organ transplantation is paving opportunities for growth of the stem cell therapy.

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Moreover, expanding pipeline and development of drugs for new applications are driving growth of the global stem cells market. Growing research activities focused on augmenting the application array of stem cell will also widen the horizon of stem cell market. Researchers are consistently trying to develop novel methods for creating human stem cell in order to comply with the rising demand for stem cell production to be used for disease management.

Development of Advanced Treatment Method Augmenting Market Growth

Lately, various new studies, development of novel therapies, and research projects have come into light in the global stem cell therapy market. Some of these treatment have been by approved by regulatory bodies, while others are still in pipeline for approval of the treatment.

In March 2017, Belgian based biotech firm TiGenix has announced that its latest development- cardiac cell therapy AlloCSC-01 has reached in its phase I/II successfully. It has shown positive results. Meanwhile, the U.S. FDA has also approved the treatment method. If this therapy is well-accepted among the patients, then approximately 1.9 million AMI patients could be treated using the therapy.

Likewise, another significant development that has been witnessed is development novel stem cell based technology for treatment of multiple sclerosis (MS) and similar concerns associated with nervous system. The treatment is developed by Israel-based Kadimastem Ltd. Also, the Latest development has been granted a patent by reputed regulatory body.

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Some of the prominent companies operating in the global stem cell therapy landscape are Anterogen Co. Ltd., RTI Surgical, Osiris Therapeutics Inc., Holostem Terapie Avanzate S.r.l., JCR Pharmaceuticals Co. Ltd., MEDIPOST Co. Ltd., Pharmicell Co. Ltd., and NuVasive Inc.

Some of these firms are following various growth strategies such as mergers and acquisitions, strategic alliances, and collaborations, and product development in order to strengthen their foothold in the global market for stem cell therapy.

Dermatology Segment Holds Prominence in Stem Cell Therapy Market

Stem cell therapy, primarily is a regenerative medicine. It encourages the reparative response of damaged, dysfunctional, or diseases tissue with the help of stem cells and associated derivatives. The treatment method is replacing the conventional transplant methods.

Stem cell therapy method has wide array of application in the field of nervous system treatment, dermatology, bone marrow transplant, multiple sclerosis, osteoarthritis, hearing loss treatment, cerebral palsy, and heart failure. The method aids patients fight leukemia and similar blood related diseases.

Among all, dermatology segment is leading in the global stem cell therapy market. The segment is substantially contributing to growth of the market. Stem cell therapy reduces the after effects of general treatment for burns such as adhesion, infections, and scars among others.

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Meanwhile, rising number of patient suffering from diabetes and increase in trauma surgery cases are anticipated to accelerate the adoption of stem cell therapy in the dermatology segment.

About TMR Research

TMR Research is a premier provider of customized market research and consulting services to business entities keen on succeeding in todays supercharged economic climate. Armed with an experienced, dedicated, and dynamic team of analysts, we are redefining the way our clients conduct business by providing them with authoritative and trusted research studies in tune with the latest methodologies and market trends.

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Stem Cell Therapy Market Grows on Back of Growing Awareness Regarding Regenerative Treatment Methods - BioSpace

Stem Cell Therapy Market to Incur Rapid Extension During 2025 – Owned

Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

Know the Growth Opportunities in Emerging Markets

Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

The regional analysis covers:

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Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

About TMR Research:

TMR Research is a premier provider of customized market research and consulting services to business entities keen on succeeding in todays supercharged economic climate. Armed with an experienced, dedicated, and dynamic team of analysts, we are redefining the way our clients conduct business by providing them with authoritative and trusted research studies in tune with the latest methodologies and market trends.

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Stem Cell Therapy Market to Incur Rapid Extension During 2025 - Owned

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