Archive for the ‘Skin Stem Cells’ Category

We are thrilled to share an excerpt form Dr. Q Schulte aufm Erley's article on Plant Stem Cells. Dr. Q is an entrepreneur, scientist and founder of one of our most loved partners: Shtrands. Shtrands is a beauty industry innovator. They provide a hair care concierge service that brings you curated products and expert advice to match your hair texture, scalp condition and styling needs.

The highly competitive cosmetics industry is always looking for the next best ingredient(s) that can fight the aging process and this led to a sizable increase in the number of anti-aging products on the market. With this is coming an increased number of active ingredients developed for this category; one of these ingredients is stem cell extract.This is an ingredient that must be assessed carefully, as marketing claims often push the limits of the available science.

The concept of stem cells originated at the end of the 19th century as a theoretical postulate to account for the ability of certain tissues (blood, skin, etc.) to renew themselves for the lifetime of organisms even though they are comprised of short-lived cells. Stem cells isolation and identification happened many years later though.

Stem cells have received a fair share of attention in the public debate mostly in connection with their potential for biomedical application and therapies. While the promise of organ regeneration have captured our imagination, it has gone almost unnoticed that plant stem cells represent the ultimate origin of much of the food we eat, the oxygen we breathe, as well the fuels we burn. Thus, plant stem cells may be ranked among the most important cells for human well-being.

A stem cell is a generic cell that can make exact copies of itself (daughters) indefinitely. These daughters can remain stem cells or further undergo differentiation (2). Such that a stem cell has the ability to make specialized cells for various tissues in the body, such as heart muscle, skin tissue, and liver tissue.

Because of their self-renewal functions, stem cells are the most important cells in the skin, as they are the source for continuous regeneration of the epidermis. Stem cell cosmetics are developed based on stem cell technology, which involves using extracts or culture media of stem cells. However, cosmetics containing human stem cells or their extracts have not been released into the market due to legal, ethical, and safety concerns. Meanwhile, plant stem cells, which circumvent these problems, are highly regarded in the cosmetics industry for improving culture technology.

The EUprohibits the use of cells, tissues, or products of human origin in cosmetics; stem cell therapy for anti-aging has not been approved or been deemed safe or effective in USA by the FDA. Furthermore, its use outside of a clinical research trial (which would be listed at http://www.clinicaltrials.gov) is prohibited. Whereas the Korea Food and Drug Association has allowed the use of sources originating from stem cell media in cosmetics since 2009 (3).

So, any cosmetics marketed as containing stem cells found on US market (should) contain stem cells extracted from plants.

A major difference between animal and plant stem cells is that plant stem cells provide cells for complete organs (branches, leaves, etc.), compared with the animal stem cells, which regenerate cells restricted to one tissue type.

Plants have nowhere to run when times get tough, so they must rely on an inner body plan to generate developmental responses to environmental changes.

Research by many labs in the last decades has uncovered a set of independent stem cell systems that fulfill the specialized needs of plant development and growth in four dimensions. In some long-lived plants, such as trees, plant stem cells remain active over hundreds or even thousands of years, revealing the exquisite precision in the underlying control of proliferation, self-renewal and differentiation.

There is some confusion around the term stem cell due to the marketing verbiage used by the cosmetic companies. In topical cosmetics the formulations dont contain stem cells straight out of the plants. They are actually a range ofplant stem cell extracts, which are manufactured using a cell culture technology.This technology consists of many and complicated methods that should ensure growth of plant cells, tissues or organs in the environment with a microbe-free nutrient. The plant cell technologyallows synthesis of the biologically active substances that exist in plants, but are not commonly available in natural environment or are difficult to obtain by chemical synthesis.

The extracts obtained through this technology from the plant stem cells are currently used for production of both common or professional care cosmetics (4).

The beneficial apple properties are known for centuries. Apples are cultivated today only for their taste, but earlier the main criterion of the type selection was the shelf life of the fruits.

One of such apple-tree types isUttwiler Spatlauberwhich is growing in Switzerland. This is a type cultivated solely due to a possible long-time storage of fruits, which remain fresh even for several months.Some trees come from the plant cutting sets planted during the 18th century!!!

The stem cell extracts are made in 2 main steps: first, the tissue material is obtained from apples (collected from a cut surfaces of the apples). Secondly, the material is going through a complicated biotechnological process to make the stem cell extracts that contains certain active ingredients. These are actually the ingredients used in formulations marketed as containing stem cells (5).

Swiss biotech company Mibelle Biochemistry created the product named PhytoCellTecTMMalus Domestica, that is a liposomal formulation (extract) derived from the stem cells of the Uttwiler Spatlauber apples. The company has published in vitro experiments done with hair follicles that showed the ability of theUttwiler Spatlauberstem cell extract to delaying of the tissue atrophy process (6); this ingredient delays hair aging.

At Teadora, we chose to includeMibelle'sPhytoCellTecTM Argan Plant Stem Cells in our ButterandBrazilian Glow Oiland here are the details from Mibelle that helped to convince us this ingredient was a must have companion to the huge list of active superfruits we crafted into our products, read on, it's pretty cool:

Deep-Seated Rejuvenation of the Skin:In order to maintain the skin in a healthy condition,cutaneous tissue is being continuously regenerated.This regenerative capacity relies on adult stem cells inthe skin. While considerable research has been done onepidermal stem cells, dermal stem cells were identifiedonly in 2009. The dermis is the middle layer of the skinand gives it tensile strength and elasticity, therefore it isalso the site where wrinkles originate.

PhytoCellTec Argan was developed to improvethe regenerative capacity of dermal stem cells therebyachieving deep-seated rejuvenation of the skin.

PhytoCellTec Argan is a powder based on stem cellsof the argan tree, one of the oldest tree species in theworld.In order to evaluate which active ingredient effectivelypromotes dermal stem cell activity, a stable humandermal papilla cell line was used as a new test system:stem cell activity is assessed based on the expression ofthe Sox2 gene, which is an established stem cell marker.Furthermore, the characteristic property of stem cells togrow in three-dimensional spherical colonies serves asa second observable indicator of stem cell viability inthis assay.

Clinical studies performed on healthy volunteers showedthat PhytoCellTec Argan:

effectively stimulates the regeneration of dermalconnective tissue, thereby increasing skin density

helps the skin to regain its firmness

significantly reduces wrinkle depth in crows feet area.

PhytoCellTec Argan is the very first active ingredientthat is capable of both protecting and vitalizing humandermal stem cells. This will not only help to acceleratethe skins natural repair process but also fights skin agingright at the root. Here are some of the amazing benefits:

Vitalizes and protects dermal stem cells Reduces wrinkles Tightens and tones skin tissues Increases skin firmness and density Deep-seated rejuvenation of the skinFirst cosmetic active with proven results forprotecting and vitalizing dermal stem cells

Continued here:
Benefits of Plant Stem Cells for Skin & Hair | Teadora

Dublin, March 10, 2020 (GLOBE NEWSWIRE) -- The "Cell Therapy - Technologies, Markets and Companies" report from Jain PharmaBiotech has been added to ResearchAndMarkets.com's offering.

The cell-based markets was analyzed for 2018, and projected to 2028. The markets are analyzed according to therapeutic categories, technologies and geographical areas. The largest expansion will be in diseases of the central nervous system, cancer and cardiovascular disorders. Skin and soft tissue repair as well as diabetes mellitus will be other major markets.

The number of companies involved in cell therapy has increased remarkably during the past few years. More than 500 companies have been identified to be involved in cell therapy and 309 of these are profiled in part II of the report along with tabulation of 302 alliances. Of these companies, 170 are involved in stem cells.

Profiles of 72 academic institutions in the US involved in cell therapy are also included in part II along with their commercial collaborations. The text is supplemented with 67 Tables and 25 Figures. The bibliography contains 1,200 selected references, which are cited in the text.

This report contains information on the following:

The report describes and evaluates cell therapy technologies and methods, which have already started to play an important role in the practice of medicine. Hematopoietic stem cell transplantation is replacing the old fashioned bone marrow transplants. Role of cells in drug discovery is also described. Cell therapy is bound to become a part of medical practice.

Stem cells are discussed in detail in one chapter. Some light is thrown on the current controversy of embryonic sources of stem cells and comparison with adult sources. Other sources of stem cells such as the placenta, cord blood and fat removed by liposuction are also discussed. Stem cells can also be genetically modified prior to transplantation.

Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering and regenerative medicine. Pharmaceutical applications of stem cells including those in drug discovery are also described. Various types of cells used, methods of preparation and culture, encapsulation and genetic engineering of cells are discussed. Sources of cells, both human and animal (xenotransplantation) are discussed. Methods of delivery of cell therapy range from injections to surgical implantation using special devices.

Cell therapy has applications in a large number of disorders. The most important are diseases of the nervous system and cancer which are the topics for separate chapters. Other applications include cardiac disorders (myocardial infarction and heart failure), diabetes mellitus, diseases of bones and joints, genetic disorders, and wounds of the skin and soft tissues.

Regulatory and ethical issues involving cell therapy are important and are discussed. Current political debate on the use of stem cells from embryonic sources (hESCs) is also presented. Safety is an essential consideration of any new therapy and regulations for cell therapy are those for biological preparations.

Key Topics Covered

Part I: Technologies, Ethics & RegulationsExecutive Summary 1. Introduction to Cell Therapy2. Cell Therapy Technologies3. Stem Cells4. Clinical Applications of Cell Therapy5. Cell Therapy for Cardiovascular Disorders6. Cell Therapy for Cancer7. Cell Therapy for Neurological Disorders8. Ethical, Legal and Political Aspects of Cell therapy9. Safety and Regulatory Aspects of Cell Therapy

Part II: Markets, Companies & Academic Institutions10. Markets and Future Prospects for Cell Therapy11. Companies Involved in Cell Therapy12. Academic Institutions13. References

For more information about this report visit https://www.researchandmarkets.com/r/bzimne

Research and Markets also offers Custom Research services providing focused, comprehensive and tailored research.

Read more:
Cell Therapy Insights Report, 2018-2028: Markets, Technologies, Ethics, Regulations, Companies & Academic Institutions - Benzinga

I feel like a sacrificial lamb, or an acceptable casualty. When a politician or scientist or couch expert says, Its only the old and ill that die, they are talking about me. The coronavirus threat has changed my identity from that of a father, husband, son, friend, pastor, alcoholic with two years sobriety and a slew of chips to prove it, to that of a comforting statistic. My new identity may soon be summed up on the news when they say, Its OK. He had underlying health problems.

Im not complaining about those who are young and healthy. When I consider my children and my wife, I, like many of you, thank God for their health. Three months ago, I would have said the same thing about my health. Unfortunately, that changed in January when I finally decided to go to the doctor and have the little red spots that had formed all over my skin, along with the new bruises that showed up daily, looked at. I found out that the blood platelets that keep all of us from bleeding to death had decided to take a vacation from my body. Most people have anywhere from 150,000 to 450,000 platelets per microliter of blood. Mine were at 4,000, and there was a danger of blood seeping into my brain, ending my life.

The first time I was in the hospital, I was there for eight days. They ran tests and stuck me with needles, capping it all off with a bone-marrow biopsy. I was diagnosed with a rare blood disorder called aplastic anemia. To put it simply, my stem cells are under siege, making it difficult for my body to produce platelets, as well as red and white blood cells. I have been hospitalized around 45 days since that diagnosis. Im actually writing this article from a hospital room. Theres a truly stunning view of the hospital roof outside my window. With no white blood cells, my immune system is completely compromised, and every little infection that normal people fight off without even noticing brings me back to the hospital again.

The only cure for me is to have a bone-marrow transplant. The problem is, even though there are several matches for my transplant, in order for the transplant to go as well as possible I have to be free of infections, viruses and other diseases. Only then can I be admitted to the University of Minnesotas Blood and Bone Marrow Transplant Center. Last month this didnt bother me. Now, though, the coronavirus is coming up behind me, daring me to wait longer.

If all goes well, Ill be discharged from the hospital and self-quarantined to my house with a bag full of antibiotics and other drugs. Sadly, Ive been told that the other things I need to stay healthy and get to my transplant are gone. The hand sanitizer, the antibacterial wipes, the masks that my family should be wearing, and the N95 respirators that I need to wear are nowhere to be found someone actually stole a box of masks from outside my hospital room. Many of these items have been snatched up by the same folks who thank God they are not me.

Again, dont get me wrong. I am grateful for those who are young and healthy. They should be thankful, for they are blessed. I just pray that when they give that thanks, they remember those of us who are terrified by what is coming. We are not simply a means to calm peoples fears; we are people who deserve compassion, mercy and dignity. I ask simply that you pray for the old and sick that they might get through this, that you leave a little hand sanitizer on the shelves for us, and for goodness sake, dont take the N95 respirators from my hospital room. I need them because my family, who knows that I am more than a comforting statistic, needs me. They need the man who loves them dearly and desperately wants to continue to be their father and husband.

Howard Baird lives in Maple Grove.

Visit link:
OPINION EXCHANGE | Amid the coronavirus threat: A plea on behalf of the old and sick - Minneapolis Star Tribune

derma aesthetics are proud to announce the launch of the Australasian Academy of Corneotherapy in Australia and New Zealand. The academy has been established to provide cutting edge Corneotherapy and skin education to all skin therapists, with the aim of advancing the level of in-depth skin health knowledge amongst the local industry.

Simone Vescio, Founder of the Australasian Academy of Corneotherapy says, Over the past eight years we have pioneered the awareness and education of corneotherapy across Australia and New Zealand, and were thrilled to be opening up our education to the aesthetics industry in Australasia!

Throughout 2020, the AAC will be holding series of Skin Extension Education Classes with the first classes having already opened their doors and been held in Sydney and Auckland in February.

From 2020 we are opening up our educational classes, training and access to the latest information on corneotherapy to all therapists, no matter what their brand of choice may currently be. There was a lack of non-product aligned education in our local industries, and were proud to be filling this gap, said Simone.

AAC Skin Extension Education 2020 - Class Schedule:Tuesday, 17 March 2020 PerthMonday, 30 March 2020 MelbourneMonday, 18 May 2020 AlburyMonday, 27 July 2020 BrisbaneMonday, 2 November 2020 Christchurch

The AAC Skin Extension Education classes have been designed to deliver comprehensive education on some of the most important and complex subjects and skin conditions. Plus, there will also be time for Q&A and further learning from your peers and educator during the day, said Simone.

Topics covered in each class include:

See the article here:
Press Release: Introducing the Australasian Academy of Corneotherapy - PRWire

There are only two northern white rhinos left on the planet, and theyre both female. Unless scientists can make a dramatic breakthrough, the entire species will die with those two individuals.

In a nondescript building just north of San Diego, California, the fight to save the northern white rhino is coming down to the wire. However, the battleground here looks less like a scene from a wildlife documentary and more akin to something out of a science fiction novel.

At the San Diego Zoo Institute for Conservation Research, an army of scientists armed with liquid nitrogen, microscopes, and ultrasound machines is working around the clock to create an unprecedented first in the conservation world: they are looking to turn frozen rhino skin cells into baby rhinos.

Its not just the science that is groundbreaking, but also the team looking to save this species. Composed mostly of women, the lab is a rarity in a field traditionally dominated by men.

Find out more about Call to Earth and the extraordinary people working for a more sustainable future

The first step in this conservation effort began more than four and a half decades ago in 1975 when scientists established the institutes Frozen Zoo. In a small room measuring no more than 36 square meters the skin cells of more than 10,000 individuals across 1,100 species sit in giant steel tanks suspended in time, frozen in liquid nitrogen.

Among the collection are the skin samples of 12 northern white rhinos. These are vital to the groups efforts because there is such a small gene pool of living northern whites.

The population has been decimated by poachers, who target rhinos because of the belief in parts of Asia that their horns can cure various ailments. The two surviving females both live under guard at the Ol Pejeta Conservancy in Kenya. Even thoughembryos have been producedin an Italian lab using eggs extracted from the pair, any future descendants from this kind of embryo would carry the genes of those two females.

That may not be enough genetic diversity to maintain a stable population. The hope is that the skin samples of those 12 individuals at the Frozen Zoo contain enough diversity to sustain the northern white species long-term.

The arduous task for these scientists is to create a rhino population from those samples.

Marlys Houck is curator of the Frozen Zoo. She graduated high school in 1979, the same year the Frozen Zoo froze its very first northern white rhino skin cell. She later joined the institute to work on the rhino project.

I was hired specifically to try to make the cells of the rhinos grow better because they were one of the most difficult to grow cell lines, she told CNN.

Since then, shes figured out how to successfully grow and freeze the skin cells of the northern white.

The impact of this work is not lost on her. Were losing species so rapidly, she said. One of the things we can do is save the living cells of these animals before its too late.

Were at the forefront of science today, she added. If we do everything right these cells should be here 50 years from now being used for purposes that we cant even imagine today.

Marisa Korody is one of the four scientists tasked with turning these frozen cells into new life. They have to reprogram the frozen skin cells into pluripotent stem cells. In laymans terms, Korody explains that stem cells can become any cell type in the body if theyre given the right signals.

The aim is to ultimately turn the stem cells into sperm and eggs. The ambitious feat has only been achieved in animals by Japanese scientists. While Korody and her team have looked to that research as a road map, she admits that doing the same with rhinos is uncharted territory. We dont really know what twists and turns we need to take in order to get from A to B, she said.

They havent even figured out how to do this in humans, she added. We have as much information as we possibly can about humans. We have a fraction of that for rhinos.

Korody says being at the forefront of this kind of science has been a dream job. This was really the first project thats trying to apply this type of science to conservation as a whole, she said.

She may spend most of her time at work looking through the lens of a microscope, but her mind is always on the final goal for the rhinos: We want to be able to put them back into the wild one day and have them living free.

Because the remaining two female northern white rhinos cant carry a pregnancy, even if the team can create embryos, the last obstacle is finding rhinos who can carry them to term.

The woman tasked with that job is Barbara Durrant. As the director of reproductive sciences, shes spent four years studying the reproductive systems of six female southern white rhinos at the institutes sister facility, the Nikita Kahn Rhino Rescue Center.

Though the rhinos at the center are a different species, Durrant says they are the closest relative to the northern white. The aim is to eventually have them be surrogates for northern white embryos.

On any given day, Durrant can be found conducting ultrasounds to help her understand each rhinos distinct reproductive cycle. In 2019, two of the centers females gave birth to southern white babies. Both were conceived via artificial insemination, giving Durrant and the teams working on the rhino project hope for the future.

Durrant believes one reason the project works so well is because there are so many women involved. Women are naturally collaborative with each other, she said. Because we have so many obstacles along the way and challenges and setbacks, we support each other and we have sympathy for each other.

Read: Rare bird brought back from extinction in the wild

Houck says women tend to be naturally nurturing. The cells are living little organisms that were growing and tending almost every day, and I think women are drawn to taking care of something and growing it into something more.

Its wonderful leading a team of women, and I really think theyre changing the world, she added. People are going to look back and see it was this amazing group of women who quietly, unrecognized, work at this and just get better and better.

Read the rest here:
Meet the women racing to save the northern white rhino from extinction in San Diego - KTLA

Dr. Meng-Lun Hsieh and Dr. Andrew Dean Shubin were married March 7 at the Warwick Melrose Hotel in Dallas. The Rev. Beth Dana, a Unitarian Universalist minister, officiated.

The bride, 33, who goes by Meng, is a fourth-year medical school student at Michigan State University, from which she also received a doctorate in biochemistry. She graduated from Williams College.

She is a daughter of Huey-Jen Liaw and Jyh-Cheng Hsieh of San Diego. The brides father is a research scientist at Sheatech, a biotech company in San Diego. Her mother, a stay-at-home parent, was a history teacher at Taibei High School, a private school in Taipei.

The groom, also 33, is a second-year general surgery resident at the University of Texas Southwestern Medical Center in Dallas. He graduated magna cum laude from the University of Washington, and received a doctorate in biomedical engineering from the University of Rochester, from which he also received a medical degree.

He is a son of Carol E. Shubin and Andy N. Shubin of Vancouver, Wash. His father retired as a photography teacher at Shahala Middle School in Vancouver. The grooms mother retired as a special-education teacher at Burton Elementary School in Vancouver.

The couple met a decade ago at the National Institutes of Health in Bethesda, Md., as post-baccalaureate research fellows, she focusing on a virus that infects bacteria, and he on skin stem cells. In April 2016, they met again at a conference for physician scientists in Chicago, and began dating long-distance.

Read this article:
Meng Hsieh, Andrew Shubin - The New York Times

(CNN) -

There are only two northern white rhinos left on the planet, and they're both female. Unless scientists can make a dramatic breakthrough, the entire species will die with those two individuals.

In a nondescript building just north of San Diego, California, the fight to save the northern white rhino is coming down to the wire. However, the battleground here looks less like a scene from a wildlife documentary and more akin to something out of a science fiction novel.

At the San Diego Zoo Institute for Conservation Research, an army of scientists armed with liquid nitrogen, microscopes, and ultrasound machines is working around the clock to create an unprecedented first in the conservation world: they are looking to turn frozen rhino skin cells into baby rhinos.

It's not just the science that is groundbreaking, but also the team looking to save this species. Composed mostly of women, the lab is a rarity in a field traditionally dominated by men.

Find out more about Call to Earth and the extraordinary people working for a more sustainable future

The first step in this conservation effort began more than four and a half decades ago in 1975 when scientists established the institute's "Frozen Zoo." In a small room measuring no more than 36 square meters the skin cells of more than 10,000 individuals across 1,100 species sit in giant steel tanks suspended in time, frozen in liquid nitrogen.

Among the collection are the skin samples of 12 northern white rhinos. These are vital to the group's efforts because there is such a small gene pool of living northern whites.

The population has been decimated by poachers, who target rhinos because of the belief in parts of Asia that their horns can cure various ailments. The two surviving females both live under guard at the Ol Pejeta Conservancy in Kenya. Even thoughembryos have been producedin an Italian lab using eggs extracted from the pair, any future descendants from this kind of embryo would carry the genes of those two females.

That may not be enough genetic diversity to maintain a stable population. The hope is that the skin samples of those 12 individuals at the Frozen Zoo contain enough diversity to sustain the northern white species long-term.

The arduous task for these scientists is to create a rhino population from those samples.

Marlys Houck is curator of the Frozen Zoo. She graduated high school in 1979, the same year the Frozen Zoo froze its very first northern white rhino skin cell. She later joined the institute to work on the rhino project.

"I was hired specifically to try to make the cells of the rhinos grow better because they were one of the most difficult to grow cell lines," she told CNN.

Since then, she's figured out how to successfully grow and freeze the skin cells of the northern white.

The impact of this work is not lost on her. "We're losing species so rapidly," she said. "One of the things we can do is save the living cells of these animals before it's too late."

"We're at the forefront of science today," she added. "If we do everything right ... these cells should be here 50 years from now being used for purposes that we can't even imagine today."

Marisa Korody is one of the four scientists tasked with turning these frozen cells into new life. They have to reprogram the frozen skin cells into pluripotent stem cells. In layman's terms, Korody explains that "stem cells can become any cell type in the body if they're given the right signals."

Read: Former war zones turn into wildlife 'paradise'

The aim is to ultimately turn the stem cells into sperm and eggs. The ambitious feat has only been achieved in animals by Japanese scientists. While Korody and her team have looked to that research as a road map, she admits that doing the same with rhinos is uncharted territory. "We don't really know what twists and turns we need to take in order to get from A to B," she said.

"They haven't even figured out how to do this in humans," she added. "We have as much information as we possibly can about humans. We have a fraction of that for rhinos."

Korody says being at the forefront of this kind of science has been a dream job. "This was really the first project that's trying to apply this type of science to conservation as a whole," she said.

She may spend most of her time at work looking through the lens of a microscope, but her mind is always on the final goal for the rhinos: "We want to be able to put them back into the wild one day and have them living free."

Because the remaining two female northern white rhinos can't carry a pregnancy, even if the team can create embryos, the last obstacle is finding rhinos who can carry them to term.

The woman tasked with that job is Barbara Durrant. As the director of reproductive sciences, she's spent four years studying the reproductive systems of six female southern white rhinos at the institute's sister facility, the Nikita Kahn Rhino Rescue Center.

Though the rhinos at the center are a different species, Durrant says they are the closest relative to the northern white. The aim is to eventually have them be surrogates for northern white embryos.

On any given day, Durrant can be found conducting ultrasounds to help her understand each rhino's distinct reproductive cycle. In 2019, two of the center's females gave birth to southern white babies. Both were conceived via artificial insemination, giving Durrant and the teams working on the rhino project hope for the future.

Durrant believes one reason the project works so well is because there are so many women involved. "Women are naturally collaborative with each other," she said. "Because we have so many obstacles along the way and challenges and setbacks, we support each other and we have sympathy for each other."

Read: Rare bird brought back from extinction in the wild

Houck says women tend to be naturally nurturing. "The cells are living little organisms that we're growing and tending almost every day, and I think women are drawn to taking care of something and growing it into something more."

"It's wonderful leading a team of women, and I really think they're changing the world," she added. "People are going to look back and see it was this amazing group of women who quietly, unrecognized, work at this and just get better and better."

Follow this link:
Meet the women racing to save the northern white rhino from extinction - KAKE

I consider my nighttime skincare routine as me time. I always double cleanse; first with Bioderma Sensibio H20 Micellar Water, 10.80, and then I use Rodial Pink Diamond Cleansing Balm, 55, and massage it into my skin. Its a really light and gentle balm that has enough slip for me to be able to easily move it around my face, and it helps dissolve any left over SPF and make-up while also getting rid of the days dirt and grime.

After a good 10-15 minute cleanse, I sweep Pestle and Mortar NMF Lactic Acid Toner, 28, around my face. Lactic acid works underneath the skin, helping to get rid of dead skin cells without dehydrating my skin. Then I press SkinCeuticals HA Intensifer, 90, into my skin to help increase hydration levels.

I use my final product once Im in bed; Decleors Bigarade Neroli Night Balm, 45, which I do a deep pressure massage with and then I finish off using a gua sha to help drain any fluids and relax the muscles.

Read this article:
3 skin experts share their morning and evening skincare routines - Stylist Magazine

EVERY 20 minutes, someone in the UK is diagnosed with a form of blood cancer, such as leukaemia or lymphoma. It's the fifth most common cancer, and the third biggest cause of cancer deaths.

Yet warning signs of blood cancers can be so unlike those of other cancers, that it's often diagnosed at a very late stage. Research by the blood cancer charity Bloodwise (bloodwise.org.uk) found more than a third of people with blood cancer had to visit their GP three or more times with symptoms before being referred to hospital. This makes it the worst performing cancer in terms of early diagnosis, the charity says.

Why is it so difficult to spot? Well, blood cancers, which stop blood stem cells from working normally and can make you weak and prone to infections, have three main types with many different variations. These variations have numerous diverse symptoms, which can often be mistaken for other less serious conditions.

"Not all signs of blood cancer are easily identifiable, or are associated with typical symptoms of cancer, such as a lump or abnormal mole," explains haematologist Dr Manos Nikolousis, a medical consultant with the blood cancer charity DKMS (dkms.org.uk).

"Blood cancer often presents in ways which are most commonly associated with unrelated and less serious illnesses, like a cold or flu. In other circumstances, patients notice a change in their body which they can't quite put their finger on."

One of the treatments for blood cancer is a stem cell transplant that restores blood-forming stem cells in patients who've had theirs destroyed by very high doses of chemotherapy and/or radiotherapy. But Nikolousis points out that only one in three blood cancer patients who need a transplant will find a matching blood stem cell donor in their family. The remaining two-thirds have to rely on an unrelated donor, which significantly reduces their chance of finding a crucial match.

"The more people who register as potential blood stem cell donors, the better the chances of finding a donor for someone in need," stresses Nikolousis, who points out that potential donors, who should be healthy and aged between 17-55, just need to sign up with DKMS online.

The charity will send a screening kit to obtain a swab from inside the donor's cheeks. If they then prove to be a match for someone, 90 per cent of donations are done by collecting blood and harvesting stem cells from it. The process is simple and could potentially save lives.

What else do you need to know about blood cancer? Here, Nikolousis outlines some blood cancer symptoms and warning signs...

1. Back pain

Musculoskeletal pain in muscles, joints, tendons, bones or structures that support the limbs, neck or back.

2. Bruising or bleeding

One of the most common symptoms associated with blood cancer is bruising, and/or bleeding. The frequency and lasting impact of bruising can be a key warning sign of blood cancer, so it's important to book an appointment with your GP if this develops.

3. Fatigue

Unexplained and persistent tiredness is one of the biggest telltale signs of blood cancer. People who have cancer-related fatigue find it incredibly challenging to complete simple tasks that we tend to take for granted.

4. Swollen lymph nodes/glands

The lymph nodes are small lumps of tissue that contain white blood cells. When inflamed, they can be felt as lumps under the skin; most commonly in the neck, armpit or groin area.

5. Headaches

There may be new headaches that feel different to headaches previously experienced. They're likely to occur frequently and be severe and long-lasting. They also often appear alongside other physical symptoms associated with blood cancer.

6. Stomach discomfort

Persistent abdominal discomfort, presenting as a sharp pain or a sense of feeling full.

7. Numbness in the hands or feet

This can be described as a feeling of pins and needles/numbness in the feet that moves up to the legs, or from fingers into the arms.

8. Heart palpitations

This can feel like a fluttering, a sudden thump or a fast pounding sensation in the chest. It can also be felt in the neck or ear when lying down.

9. Loss of concentration

There may be a prolonged or recurring inability to concentrate or focus, and people may describe this as feeling mentally drained or dizzy.

10. Sleep problems

Blood cancer patients may have continuous trouble falling asleep or staying asleep. This might result in a restless night's sleep causing a feeling of extreme tiredness.

11. Itchy skin

Persistent, irritable and itchy skin. This may be experienced all over the body, or in isolated spots. Again, this symptom often appears alongside other physical symptoms associated with blood cancer.

Get it checked

These symptoms are very common and don't automatically mean you have cancer. But if you notice any unusual or ongoing changes, it's always best to see your doctor and get checked out sooner rather than later.

Link:
Blood cancer symptoms everybody needs to know - The Irish News

Painless non surgical skin tightening procedures are now available and very popular for people who don't want to go through surgery in order to look better. Call LifeGaines in Boca Raton for more information about "Thread Lifts."

Boca Raton, Florida--(Newsfile Corp. - March 5, 2020) - Thread Lifts are a simple, painless procedure used for skin tightening on the face, neck, or anywhere else on the body. As popular and effective as Botox is, it simply doesn't have the ability to lift the skin. In the past, there hasn't been a great way to produce lifting results without surgery.

Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view an enhanced version of this image, please visit:https://orders.newsfilecorp.com/files/6848/53186_lifegaines_orig.jpg

Non-surgical aesthetics are in high demand and ThreadLifts, new to the United States, have the ability to produce skin that is instantly lifted and tightened.

This procedure uses no cuts or incisions, only injections. Threads are needles that are pre-loaded with PDO thread. The whole needle is inserted in the tissue at the sub-dermal level, along the surface of the skin and then the needle is pulled out. Threads can be used nearly anywhere on the body, but they are especially effective on the neck and jawline.

What is PDO?

Polydioxanone (PDO) sutures have been used for surgical procedures for many decades. It is one of the safest materials to implant in the body. PDO is completely dissolvable and your skin fully absorbs it within 4 to 6 months, leaving no scar tissue behind. This is especially effective when it's used together with chemical peels, Botox, and fillers to effect a patient's entire facial structure, remove sunspots and other conditions.

It is a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. They are also effective on the breasts, buttocks and upper arms, areas that are prone to sagging due to weight loss, aging, pregnancy and childbirth or poor muscle tone.

Thread Lifts are a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. Results from threads generally last between 12 months to several years depending on the area that has been treated, how many threads are used and what kinds of threads are used in each needle.

Amy Steffey, is a Licensed Nurse Practitioner with LifeGaines Medical and Aesthetics, and with Thread Liftsprocedure, she helps Boca Raton residents regain their confidence with rejuvenating procedures and body contouring.

Amy Steffey works at LifeGaines, which is one of the most highly respected Age Management Medical teams in South Florida. Age Management Medicine pioneer Dr. Richard Gaines is the founder of LifeGaines, and he has years of experience specializing in Hormone Replacement Therapy, Sexual Wellness, Platelet-rich Plasma, Stem Cells, Aesthetics, and Advanced Age Management protocols.

LifeGaines is located at 3785 N Federal Hwy #150, Boca Raton, FL 33431.

Call 561-295-9007 Today to Schedule a Consultation with Amy Steffey at LifeGaines Medical & Aesthetics Center in Boca Raton.

Related Images

people-who-dont-want-to-do-surgery.jpg People who don't want to do surgery could consider a non-surgical way of tightening skin. Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view the source version of this press release, please visit https://www.newsfilecorp.com/release/53186

Read the original post:
Non Surgical Aesthetics Are in High Demand, So Thread Lifts Are a New Way to Achieve Instant Skin Tightening - Yahoo Finance

1 Molecular missing link: Research explains why some creams cause a skin rash

Allergic skin reactions can be caused by many different chemical compounds found in skincare creams, cosmetics and other topical consumer products, but how they trigger the reaction has remained somewhat mysteriousuntil now.

New research that suggests the way some chemicals displace natural fat-like molecules known as lipids in skin cells may explain how many common ingredients trigger allergic contact dermatitis.

The breakthrough could help stem soaring cases of rashes, lumps, blisters, itchy eyes and facial swellings. It has been dubbed the molecular missing link because it might have brought a new way to treat the condition.

Currently, the only way to stop allergic contact dermatitis is to identify and avoid coming into contact with the chemical that causes the reaction.

Most allergies are attributed to proteins or synthetically produced peptide antigens that set off the immune system.

A new study from the Hamamatsu University School of Medicine has discovered that docosahexaenoic acid (DHA) could potentially be an important component in creating more efficient lip care products.

The university collaborated with Kose Corporations research laboratories in order to better understand the molecular profile of the lip area.

The study highlighted it was especially important to study the lip in closer detail as it was one of the major targets of cosmetics.

Its important to under the molecular profile specific to human lips to discover the intrinsic ingredients for lip cosmetics.

To gain a better understanding, the team aimed to map out the human lip using imaging mass spectrometry to gather insight into its lipid distribution.

Researchers at Kao Corporation have developed a method to predict odour intensity, a skill previously dependent on the experience of perfumers.

The development of the technology was the result of a project by the firms Sensory Science Research Laboratory, which compiled a database of 314 commonly used fragrance ingredients.

The database of olfaction characteristics allowed scientists to develop a method that can predict the odour intensity based on the concentration of perfumery raw materials (PRMs) present in a gas sample.

Data were obtained from an evaluation testing performed by 18 perfumers and researchers, who scored the intensity of samples emitted from a fragrance diluter with different gas concentrations.

Based on evaluations of those results, the team managed to visualise the relationship between gas concentration and odour intensity.

Singapore biotech start-up Insectta is rearing black soldier flies on its urban farm to produce a more sustainable and purer chitosan for the cosmetics industry.

The company, which claims to have the first insect farm in Singapore, raises black soldier flies that feed on food waste before being converted into viable materials such as chitosan.

It is a useful material that has many functions. For cosmetics, it has antioxidant, antimicrobial and wound-healing properties. Additionally, it can enhance penetration into skin.

Chitosan is conventionally sourced from shrimp and crab. However, the company believes using insects is cleaner and more sustainable.

These flies are not pests, they don't bite. They are native to Singapore and not an invasive species. They feed on food waste which would otherwise go into the incinerator. We're trying to promote a circular economy, said Chua Kai Ning, chief marketing officer of Insectta.

The flies on the farm, which are housed in a small room, can consume around 7.5 tonnes of food waste a month.

Experts in Australia have been urging the public to carry on using sunscreen even though a widely publicised study has shown chemicals can be absorbed from some products into the bloodstream.

US Food and Drug Administration scientists found that sunscreen users might be taking in more of the active ingredients into our blood, far beyond regulatory thresholds.

They tested six of the main active ingredients in sunscreen lotions, sprays and pumps, revealing quantities of sunscreen chemicals in the blood high enough for the products to have to undergo additional FDA safety studies.

This happens when formulations surpass a threshold that requires them to be taken for further testing. The chemicals studied were avobenzone, oxybenzone, octocrylene, ecamsule, homosalate, octisalate and octinoxate.

The researchers stress the findings do not mean that sunscreens are unsafe, merely that more research is needed. The FDA will now conduct more research to determine the maximum levels of sunscreen ingredients that are safe to use.

Original post:
Skin science: Top five stories on cosmetics science and formulation - CosmeticsDesign-Asia.com

Article In Brief

A unique molecular structureevident in induced pluripotent stem cells taken from people with young-onset Parkinson's diseasesuggests that the defects may be present throughout patients' lives, and that they could therefore be used as diagnostic markers.

Induced pluripotent stem cells (iPSCs) taken from patients with young-onset Parkinson's disease (YOPD) and grown into dopamine-producing neurons displayed a molecular signature that was corrected in vitro, as well as in the mice striatum, by a drug already approved by the US Food and Drug Administration (FDA), a study published in the January 27 online edition of Nature Medicine found.

Although the patients had no known genetic mutations associated with PD, the neurons grown from their iPSCs nonetheless displayed abnormally high levels of soluble alpha-synucleina classic phenotype of the disease, but one never before seen in iPSCs from patients whose disease developed later in life. Surprisingly, for reasons not yet understood, the cells also had high levels of phosphorylated protein kinase C-alpha (PKC).

In addition, the cells also had another well-known hallmark of PD: abnormally low levels of lysosomal membrane proteins, such as LAMP1. Because lysosomes break down excess proteins like alpha-synuclein, their reduced levels in PD have long been regarded as a key pathogenic mechanism.

When the study team tested agents known to activate lysosomal function, they found that a drug previously approved by the FDA as an ointment for treating precancerous lesions, PEP005, corrected all the observed abnormalities in vitro: it reduced alpha-synuclein and PKC levels while increasing LAMP1 abundance. It also decreased alpha-synuclein production when delivered to the mouse striatum.

Unexpectedly, however, PEP005 did not work by activating lysosomal function; rather, it caused another key protein-clearing cellular structure, the proteasome, to break down alpha-synuclein more readily.

The findings suggest that the defects seen in the iPSCs are present throughout patients' lives, and that they could therefore be used as diagnostic markers. Moreover, the drug PEP005 should be considered a potentially promising therapeutic candidate for YOPD and perhaps even for the 90 percent of PD patients in whom the disease develops after the age of 50, according to the study's senior author, Clive Svendsen, PhD, director of the Cedars-Sinai Board of Governors Regenerative Medicine Institute and professor of biomedical sciences and medicine at Cedars-Sinai.

These findings suggest that one day we may be able to detect and take early action to prevent this disease in at-risk individuals, said study coauthor Michele Tagliati, MD, FAAN, director of the movement disorders program and professor of neurology at Cedars-Sinai Medical Center.

But the study still raises questions regarding the biological mechanisms, and certainly does not warrant off-label prescribing of PEP005 at this time, said Marco Baptista, PhD, vice president of research programs at the Michael J. Fox Foundation, who was not involved with the study.

Repurposing PEP005 is a long way away, Dr. Baptista said. This is not something that neurologists should be thinking about prescribing or recommending to their patients.

Accumulation of alpha-synuclein has been seen in iPSC-derived dopaminergic cultures taken from patients with known genetic defects, but such defects account for only about 10 percent of the PD population. In those without known mutations, on the other hand, no defects in iPSC-derived dopamine-producing neurons have been seen. Until now, however, such studies had been conducted only in patients who had developed PD after age 50.

My idea was why to look in young-onset patients, said Dr. Svendsen.

The idea paid off more richly than he expected. We were shocked to find a very, very prominent phenotype, a buildup of alpha-synuclein, in the neurons of these patients who are genetically normal, Dr. Svendsen said. None of the controls had a buildup of synuclein, and all but one of the early PD patients had a twofold increase in it.

The signature is so consistent, he said, that it offers a natural model that can be interrogated to further understand its workings.

Because high levels of PKC were also seen, Dr. Svendsen said, We picked a bunch of drugs known to reduce PKC. We found one, PEP005, which is actually extracted from the milkweed plant, and it completely reduced synuclein levels almost to normal in dopaminergic neurons. And it also increased dopamine levels in those cells, so we got two for one.

After observing the effects of PEP005 in vitro, We put it into the mouse brain and found it reduced synuclein in vivo, Dr. Svendsen said. But we had to infuse it right into the brain. We're now trying to work out how to get it across the blood-brain barrier more efficiently.

To determine how PEP005 lowers cellular levels of alpha-synuclein, his group tested whether it was activating the lysosome, but found to their surprise that it did not do this until after the synuclein had already been degraded.

Then we asked whether it could be the proteosome, which also breaks down proteins but normally doesn't break down synuclein, Dr. Svendsen said. But when we applied PEP005, it did activate the proteasome. So we think that might be the mechanism.

Because the drug is currently applied externally, Dr. Svendsen said, the next step will be to see if it crosses the blood-brain barrier when applied to the skin of mice, and whether that results in a lowering of synuclein levels in dopaminergic neurons.

Justin Ichida, PhD, the Richard N. Merkin assistant professor of stem cell biology and regenerative medicine at the USC Keck School of Medicine, said the findings are quite important in the field. The potential diagnostic tools they made could be important in clinical care. And identifying a drug that may very effectively reverse the disease in neurons is a very important discovery.

He wondered, however, whether the increase in alpha-synuclein is truly specific to Parkinson's neurons or if it would also be seen in iPSC neurons from patients with Alzheimer's disease or amyotrophic lateral sclerosis.

I wonder if alpha-synuclein accumulating is a sign of PD in a dish or is a consequence of neurodegeneration or impaired protein degradation in general, Dr. Ichida said. That's a key question if you want to use this molecular signature as a diagnostic tool.

He also questioned if proteins other than alpha-synuclein, such as tau, would also be seen to accumulate in the iPSCs of YOPD patients.

If one of the protein-clearance mechanisms in the cell is working poorly, you would imagine that other things would also accumulate, Dr. Ichida said.

In response, Dr. Svendsen said that while some proteins other than alpha-synuclein were reported in the paper at increased levels, We did not look at tau specifically, but are in the process of looking right now. It could be that synuclein and some other proteins are somehow altered to evade them from being degraded by the lysosome, or that there is a general lysosomal problem.

Patrik Brundin, MD, PhD, director of the Center for Neurodegenerative Science and Jay Van Andel Endowed Chair at Van Andel Research Institute in Grand Rapids, MI, called the paper very interesting and thought-provoking. If these findings hold up, they could shift our understanding of young-onset PD. They imply that there is a strong genetic component that has not been picked up in prior genetic studies.

Dr. Brundin said he would like to see the results replicated in another lab using different sets of reagents. It is so intriguing and rather unexpected that one wonders if the observations really apply, as the study states, to 95 percent of all YOPD.

He also questioned whether all the young-onset PD patients are similar. Clearly the iPSCs studied here are not monogenetic PD, so they must be very diverse genetically and still all have the same alpha-synuclein change.

Dr. Brundin also asked why the abnormalities seen in YOPD neurons have not previously been seen in older cases of PD. Is there a specific cutoff regarding age-of-onset when these purposed genetic differences apply? he asked.

Dr. Svendsen responded: We don't know why the YO have this phenotype or exactly what the cut off is. We have, however, looked at one adult-onset case that did not show this phenotype. Also, one of our YO cases did not show this phenotype. Thus some patients even with early onset may not have it. We are currently testing many more cases from older-onset patients.

Dr. Brundin also wanted to know whether non-dopaminergic neurons have the same deficits described in the study.

We don't know which neurons specifically have the protein deficit as we cannot do single-cell proteomics, Dr. Svendsen answered. It could be a little in all cells or a lot in a small set. Immunocytochemistry is not quantitative but showed that it is more likely a general increase in synuclein and not specific to dopaminergic neurons.

While the findings in iPSCs suggest that the abnormal levels of alpha-synuclein must be present at birth, Dr. Brundin said, I do not know how to reconcile the present findings with genetic data.

The absence of previously described mutations in the YOPD patients means only that more work must be done to uncover the genetic underpinnings, Dr. Svendsen said.

We're just at the tip of the iceberg with understanding the genome, he said. It's such a bizarrely complex beast. Perhaps there are a thousand different proteins interacting to stop the synuclein from being degraded. In 10 years, we probably will be clever enough to see it. We know it must be there. Now the genome guys will go after it.

Dr. Baptista from the Michael J. Fox Foundation said he agreed with the view that there must be genetic alterations underpinning the defects seen in the iPSCs.

Just because we call something non-genetic could simply reflect the current ignorance of the field, he said. I think the discoveries are simply difficult to make.

He added that he wished that the main comparator in the study was not healthy controls, and that there were more older-onset iPSCs to compare against YOPD patients' samples.

Dr. Svendsen said it could be that the iPSCs from older-onset patients might yet be found with additional study to display abnormalities similar to those seen in YOPD.

Right now we only see it in young onset, he said. We may need to leave the cultures longer to see in the older-onset patients. We are doing those experiments now.

Drs. Tagliati and Svendsen disclosed that an intellectual patent is pending for diagnostic and drug screening for molecular signatures of early-onset Parkinson's disease. Dr. Ikeda is a co-founder of AcuraStem Inc. Dr. Brundin has received commercial support as a consultant from Renovo Neural, Inc., Lundbeck A/S, AbbVie, Fujifilm-Cellular Dynamics International, Axial Biotherapeutics, and Living Cell Technologies. He has also received commercial support for research from Lundbeck A/S and Roche and has ownership interests in Acousort AB and Axial Biotherapeutics. Dr. Baptista had no disclosures.

Read the rest here:
Molecular Signature of Young-Onset Parkinson's Disease Is... : Neurology Today - LWW Journals

4-Mar-2020

Ingredients | Skin Care

inspira: cosmetics produces and markets high-quality, contemporary cosmetic products for individual skin care worldwide

The German company is based in Aachen and was founded in 2000. The development of outstanding and highly effective products with excellent compatibility is a matter of course for inspira: cosmetics.

The products visibly improve the appearance of the skin and let the user look in the best possible way for his/her respective age.

Now Volker Kloubert, Managing Partner of inspira: cosmetics proudly announced: "We are more than happy and feel very honoured that 3 of our entries are finalists in the Pure Beauty Global Awards and we are looking forward to the award ceremony in May in Amsterdam. Lets keep fingers crossed!

The finalist products from inspira: cosmetics reflect the broad scope of cosmetics the brand is covering.

The male scent 4MEN ONLY is nominated in the category Best Male Fragrance. A masculine composition of oriental notes, combined with woods and musk. Adventurous and very sexy! For men only. The sophisticated fragrance was created by master perfumers in Grasse/France.

Finalist in the category Best Lip Product is the Volumizing Lip Remedy, a lip care stick in stylish silver metal packaging with high quality active ingredients like hyaluronic acid, shea butter, coconut oil, spearmint oil for a fresh taste and the Peptide Complex VOLULIP than can increase the lip volume by up to 82% in 4 weeks as it stimulates the production of hyaluronic acid in the lips.

Very important: NO burning sensation, the product is smooth as silk.

Age Reboot Serum is the new holistic anti aging serum in the inspira: med range using state of the art active ingredients to protect and rejuvenate the skin.

Phyto stem cells help the skin to adapt to changing environmental conditions like heat or cold and protect the cells whereas three different hyaluronic acids smooth the skin, even out wrinkles and EGF (Epidermal Growth Factor) stimulates cell renewal.

In clinical studies the skin was rejuvenated by up to 10 years in four weeks of regular use.

See the article here:
Three of inspira: cosmetics entries are finalists in the Pure Beauty Global Awards 2020 - Cosmetics Business

Abstract

Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)like matrix to direct the conversion of MSC into functional SGs and facilitated SGs recovery in mice. By extracellular matrix differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator for SG specification. Our findings showed that Hmox1 could respond to the 3D structure activation and also be involved in MSC differentiation. Using inhibition and activation assay, CTHRC1 and Hmox1 synergistically boosted SG gene expression profile. Together, these findings indicated that biochemical and structural cues served as two critical impacts of 3D-printed matrix on MSC fate decision into the glandular lineage and functional SG recovery.

Mesenchymal stem cells (MSCs) hold great promise for therapeutic tissue engineering and regenerative medicine, largely because of their capacity for self-renewal and multipotent properties (1). However, their uncertain fate has a major impact on their envisioned therapeutic use. Cell fate regulation requires specific transcription programs in response to environmental cues (2, 3). Once stem cells are removed from their microenvironment, their response to environmental cues, phenotype, and functionality could often be altered (4, 5). In contrast to growing information concerning transcriptional regulation, guidance from the extracellular matrix (ECM) governing MSC identity and fate determination is not well understood. It remains an active area of investigation and may provide previously unidentified avenues for MSC-based therapy.

Over the past decade, engineering three-dimensional (3D) ECM to direct MSC differentiation has demonstrated great potential of MSCs in regenerative medicine (6). 3D ECM has been found to be useful in providing both biochemical and biophysical cues and to stabilize newly formed tissues (7). Culturing cells in 3D ECM radically alters the interfacial interactions with the ECM as compared with 2D ECM, where cells are flattened and may lose their differentiated phenotype (8). However, one limitation of 3D materials as compared to 2D approaches was the lack of spatial control over chemistry with 3D materials. One possible solution to this limitation is 3D bioprinting, which could be used to design the custom scaffolds and tissues (9).

In contrast to traditional engineering techniques, 3D cell printing technology is especially advantageous because it can integrate multiple biophysical and biochemical cues spatially for cellular regulation and ensure complex structures with precise control and high reproducibility. In particular, for our final goal of clinical practice, extrusion-based bioprinting may be more appropriate for translational application. In addition, as a widely used bioink for extrusion bioprinting, alginate-based hydrogel could maintain stemness of MSC due to the bioinert property and improve biological activity and printability by combining gelatin (10).

Sweat glands (SGs) play a vital role in thermal regulation, and absent or malfunctioning SGs in a hot environment can lead to hyperthermia, stroke, and even death in mammals (11, 12). Each SG is a single tube consisting of a functionally distinctive duct and secretory portions. It has low regenerative potential in response to deep dermal injury, which poses a challenge for restitution of lost cells after wound (13). A major obstacle in SG regeneration, similar to the regeneration of most other glandular tissues, is the paucity of viable cells capable of regenerating multiple tissue phenotypes (12). Several reports have described SG regeneration in vitro; however, dynamic morphogenesis was not identified nor was the overall function of the formed tissues explored (1416). Recent advances in bioprinting and tissue engineering led to the complexities in the matrix design and fabrication with appropriate biochemical cues and biophysical guidance for SG regeneration (1719).

Here, we adopted 3D bioprinting technique to mimic the regenerative microenvironment that directed the specific SG differentiation of MSCs and ultimately guided the formation and function of glandular tissue. We used alginate/gelatin hydrogel as bioinks in this present study due to its good cytocompatibility, printability, and structural maintenance in long-time culture. Although the profound effects of ECM on cell differentiation was well recognized, the importance of biochemical and structural cues of 3D-printed matrix that determined the cell fate of MSCs remained unknown; thus, the present study demonstrated the role of 3D-printed matrix cues on cellular behavior and tissue morphogenesis and might help in developing strategies for MSC-based tissue regeneration or directing stem cell lineage specification by 3D bioprinting.

The procedure for printing the 3D MSC-loaded construct incorporating a specific SG ECM (mouse plantar region dermis, PD) was shown schematically in Fig. 1A. A 3D cellular construct with cross section 30 mm 30 mm and height of 3 mm was fabricated by using the optimized process parameter (20). The 3D construct demonstrated a macroporous grid structure with hydrogel fibers evenly distributed according to the computer design. Both the width of the fibers and the gap between the fibers were homogeneous, and MSCs were embedded uniformly in the hydrogel matrix fibers to result in a specific 3D microenvironment. (Fig. 1B).

(A) Schematic description of the approach. (B) Full view of the cellular construct and representative microscopic and fluorescent images and the quantitative parameters of 3D-printed construct (scale bars, 200 m). Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Representative microscopy images of cell aggregates and tissue morphology at 3, 7, and 14 days of culture (scale bars, 50 m) and scanning electron microscopy (sem) images of 3D structure (scale bars, 20 m). PD+/PD, 3D construct with and without PD. (D) DNA contents, collagen, and GAGs of native tissue and PD. (E) Proliferating cells were detected through Ki67 stain at 3, 7, and 14 days of culture. (F) Live/dead assay show cell viability at days 3, 7, and 14. *P < 0.05.

During the maintenance of constructs for stem cell expansion, MSCs proliferated to form aggregates of cells but self-assembled to an SG-like structure only with PD administration (Fig. 1C and fig. S1, A to C). We carried out DNA quantification assay to evaluate the cellular content in PD and found the cellular matrix with up to 90% reduction, only 3.4 0.7 ng of DNA per milligram tissue remaining in the ECM. We also estimated the proportions of collagen and glycosaminoglycans (GAGs) in ECM through hydroxyproline assay and dimethylmethylene blue assay, the collagen contents could increase to 112.6 11.3%, and GAGs were well retained to 81 9.6% (Fig. 1D). Encapsulated cells were viable, with negligible cell death apparent during extrusion and ink gelation by ionic cross-linking, persisting through extended culture in excess of 14 days. The fluorescence intensity of Ki67 of MSCs cultured in 2D condition decreased from days 3 (152.7 13.4) to 14 (29.4 12.9), while maintaining higher intensity of MSCs in 3D construct (such as 211.8 19.4 of PD+3D group and 209.1 22.1 of PD3D group at day 14). And the cell viability in 3D construct was found to be sufficiently high (>80%) when examined on days 3, 7, and 14. The phenomenon of cell aggregate formation and increased cell proliferation implied the excellent cell compatibility of the hydrogel-based construct and promotion of tissue development of 3D architectural guides, which did not depend on the presence or absence of PD (Fig. 1, E and F).

The capability of 3D-printed construct with PD directing MSC to SGs in vitro was investigated. The 3D construct was dissolved, and cells were isolated at days 3, 7, and 14 for transcriptional analysis. Expression of the SG markers K8 and K18 was higher from the 3D construct with (3D/PD+) than without PD (3D/PD); K8 and K18 expression in the 3D/PD construct was similar to with control that MSCs cultured in 2D condition, which implied the key role of PD in SG specification. As compared with the 2D culture condition, 3D administration (PD+) up-regulated SG markers, which indicated that the 3D structure synergistically boosted the MSC differentiation (Fig. 2A).

(A) Transcriptional expression of K8, K18, Fxyd2, Aqp5, and ATP1a1 in 3D-bioprinted cells with and without PD in days 3, 7, and 14 culture by quantitative real-time polymerase chain reaction (qRT-PCR). Data are means SEM. (B) Comparison of SG-specific markers K8 and K18 in 3D-bioprinted cells with and without PD (K8 and K18, red; DAPI, blue; scale bars, 50 m). (C and D) Comparison of SG secretion-related markers ATP1a1 (C) and Ca2+ (D) in 3D-bioprinted cells with and without PD [ATP1a1 and Ca2+, red; 4,6-diamidino-2-phenylindole (DAPI), blue; scale bars, 50 m].

In addition, we tested secretion-related genes to evaluate the function of induced SG cells (iSGCs). Although levels of the ion channel factors of Fxyd2 and ATP1a1 were increased notably in 2D culture with PD and ATP1a1 up-regulated in the 3D/PD construct, all the secretory genes of Fxyd2, ATP1a1, and water transporter Aqp5 showed the highest expression level in the 3D/PD+ construct (Fig. 2A). Considering the remarkable impact, further analysis focused on 3D constructs.

Immunofluorescence staining confirmed the progression of MSC differentiation. At day 7, cells in the 3D/PD+ construct began to express K8 and K18, which was increased at day 14, whereas cells in the 3D/PD construct did not express K8 and K18 all the time (Fig. 2B and fig. S2A). However, the expression of ATP1a1 (ATPase Na+/K+ transporting subunit alpha 1) and free Ca2+ concentration did not differ between cells in the 3D/PD+ and 3D/PD constructs (Fig. 2, C and D). By placing MSCs in such a 3D environment, secretion might be stimulated by rapid cell aggregation without the need for SG lineage differentiation. Cell aggregationimproved secretion might be due to the benefit of cell-cell contact (fig. S2B) (21, 22).

To map the cell fate changes during the differentiation between MSCs and SG cells, we monitored the mRNA levels of epithelial markers such as E-cadherin, occludin, Id2, and Mgat3 and mesenchymal markers N-cadherin, vimentin, Twist1, and Zeb2. The cells transitioned from a mesenchymal status to a typical epithelial-like status accompanied by mesenchymal-epithelial transition (MET), then epithelial-mesenchymal transition (EMT) occurred during the further differentiation of epithelial lineages to SG cells (fig. S3A). In addition, MET-related genes were dynamically regulated during the SG differentiation of MSCs. For example, the mesenchymal markers N-cadherin and vimentin were down-regulated from days 1 to 7, which suggested cells losing their mesenchymal phenotype, then were gradually up-regulated from days 7 to 10 in their response to the SG phenotype and decreased at day 14. The epithelial markers E-cadherin and occludin showed an opposite expression pattern: up-regulated from days 1 to 5, then down-regulated from days 7 to 10 and up-regulated again at day 14. The mesenchymal transcriptional factors ZEB2 and Twist1 and epithelial transcriptional factors Id2 and Mgat3 were also dynamically regulated.

We further analyzed the expression of these genes at the protein level by immunofluorescence staining (figs. S3B and S4). N-cadherin was down-regulated from days 3 to 7 and reestablished at day 14, whereas E-cadherin level was increased from days 3 to 7 and down-regulated at day 14. Together, these results indicated that a sequential and dynamic MET-EMT process underlie the differentiation of MSCs to an SG phenotype, perhaps driving differentiation more efficiently (23). However, the occurrence of the MET-EMT process did not depend on the presence of PD. Thus, a 3D structural factor might also participate in the MSC-specific differentiation (fig. S3C).

To investigate the underlying mechanism of biochemical cues in lineage-specific cell fate, we used quantitative proteomics analysis to screen the ECM factors differentially expressed between PD and dorsal region dermis (DD) because mice had eccrine SGs exclusively present in the pads of their paws, and the trunk skin lacks SGs. In total, quantitative proteomics analyses showed higher expression levels of 291 proteins in PD than DD. Overall, 66 were ECM factors: 23 were significantly up-regulated (>2-fold change in expression). We initially determined the level of proteins with the most significant difference after removing keratins and fibrin: collagen triple helix repeat containing 1 (CTHRC1) and thrombospondin 1 (TSP1) (fig. S5). Western blotting was performed to further confirm the expression level of CTHRC1 and TSP1, and we then confirmed that immunofluorescence staining at different developmental stages in mice revealed increased expression of CTHRC1 in PD with SG development but only slight expression in DD at postnatal day 28, while TSP1 was continuously expressed in DD and PD during development (Fig. 3, A to C). Therefore, TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development.

(A and B) Differential expression of CTHRC1 and TSP1in PD and back dermis (DD) ECM of mice by proteomics analysis (A) and Western blotting (B). (C) CTHRC1 and TSP1 expression in back and plantar skin of mice at different developmental times. (Cthrc1/TSP1, red; DAPI, blue; scale bars, 50 m).

According to previous results of the changes of SG markers, 3D structure and PD were both critical to SG fate. Then, we focused on elucidating the mechanisms that underlie the significant differences observed in 2D and 3D conditions with or without PD treatment. To this end, we performed transcriptomics analysis of MSCs, MSCs treated with PD, MSCs cultured in 3D construct, and MSC cultured in 3D construct with PD after 3-day treatment. We noted that the expression profiles of MSCs treated with 3D, PD, or 3D/PD were distinct from the profiles of MSCs (Fig. 4A). Through Gene Ontology (GO) enrichment analysis of differentially expressed genes, it was shown that PD treatment in 2D condition induced up-regulation of ECM and inflammatory response term, and the top GO term for MSCs in 3D construct was ECM organization and extracellular structure organization. However, for the MSCs with 3D/PD treatment, we found very significant overrepresentation of GO term related to branching morphogenesis of an epithelial tube and morphogenesis of a branching structure, which suggested that 3D structure cues and biochemical cues synergistically initiate the branching of gland lineage (fig S6). Heat maps of differentially expressed ECM organization, cell division, gland morphogenesis, and branch morphogenesis-associated genes were shown in fig. S7. To find the specific genes response to 3D structure cues facilitating MSC reprogramming, we analyzed the differentially expressed genes of four groups of cells (Fig. 4B). The expression of Vwa1, Vsig1, and Hmox1 were only up-regulated with 3D structure stimulation, especially the expression of Hmox1 showed a most significant increase and even showed a higher expression addition with PD, which implied that Hmox1 might be the transcriptional driver of MSC differentiation response to 3D structure cues. Differential expression of several genes was confirmed by quantitative polymerase chain reaction (qPCR): Mmp9, Ptges, and Il10 were up-regulated in all the treated groups. Likewise, genes involving gland morphogenesis and branch morphogenesis such as Bmp2, Tgm2, and Sox9 showed higher expression in 3D/PD-treated group. Bmp2 was up-regulated only in 3D/PD-treated group, combined with the results of GO analysis, we assumed that Bmp2 initiated SG fate through inducing branch morphogenesis and gland differentiation (Fig. 4C).

(A) Gene expression file of four groups of cells (R2DC, MSCs; R2DT, MSC with PD treatment; R3DC, MSC cultured in 3D construct; and R3DT, MSC treated with 3D/PD). (B) Up-regulated genes after treatment (2DC, MSCs; 2DT, MSC with PD treatment; 3DC, MSC cultured in 3D construct; and 3DT, MSC treated with 3D/PD). (C) Differentially expressed genes were further validated by RT-PCR analysis. [For all RT-PCR analyses, gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with 40 cycles, data are represented as the means SEM, and n = 3].

To validate the role of HMOX1 and CTHRC1 in the differentiation of MSCs to SG lineages, we analyzed the gene expression of Bmp2 by regulating the expression of Hmox1 and CTHRC1 based on the 3D/PD-treated MSCs. The effects of caffeic acid phenethyl ester (CAPE) and tin protoporphyrin IX dichloride (Snpp) on the expression of Hmox1 were evaluated by quantitative real-time (qRT)PCR. Hmox1 expression was significantly activated by CAPE and reduced by Snpp. Concentration of CTHRC1 was increased with recombinant CTHRC1 and decreased with CTHRC1 antibody. That is, it was negligible of the effects of activator and inhibitor of Hmox1 and CTHRC1 on cell proliferation (fig. S8, A and B). Hmox1 inhibition or CTHRC1 neutralization could significantly reduce the expression of Bmp2, while Hmox1 activation or increased CTHRC1 both activated Bmp2 expression. Furthermore, Bmp2 showed highest expression by up-regulation of Hmox1 and CTHRC1 simultaneously and sharply decreased with down-regulation of Hmox1 and CTHRC1 at the same time (Fig. 5A). Immunofluorescent staining revealed that the expression of bone morphogenetic protein 2 (BMP2) at the translational level with CTHRC1 and Hmox1 regulation showed a similar trend with transcriptional changes (Fig. 5B). Likewise, the expression of K8 and K18 at transcriptional and translational level changed similarly with CTHRC1 and Hmox1 regulation (fig. S9, A and B). These results suggested that CTHRC1 and Hmox1 played an essential role in SG fate separately, and they synergistically induced SG direction from MSCs (Fig. 5C).

(A and B) Transcriptional analysis (A) and translational analysis (PD, MSCs; PD+, MSCs with 3D/PD treatment; CAPE, MSCs treated with 3D/PD and Hmox1 activator; Snpp, MSCs treated with 3D/PD and Hmox1 inhibitor; Cthrc1, MSCs treated with 3D/PD and recombinant CTHRC1; anti, MSCs treated with 3D/PD and CTHRC1 antibody: +/+, MSCs treated with 3D/PD and Hmox1 activator and recombinant CTHRC1; and /, MSCs treated with 3D/PD and Hmox1 inhibitor and CTHRC1 antibody. Data are represented as the means SEM and n = 3) (B) of bmp2 with regulation of CTHRC1 and Hmox1. (C) The graphic illustration of 3D-bioprinted matrix directed MSC differentiation. CTHRC1 is the main biochemical cues during SG development, and structural cues up-regulated the expression of Hmox1 synergistically initiated branching morphogenesis of SG. *P < 0.05.

Next, we sought to assess the repair capacity of iSGCs for in vivo implications, the 3D-printed construct with green fluorescent protein (GFP)labeled MSCs was transplanted in burned paws of mice (Fig. 6A). We measured the SG repair effects by iodine/starch-based sweat test at day 14. Only mice with 3D/PD treatment showed black dots on foot pads (representing sweating), and the number increased within 10 min; however, no black dots were observed on untreated and single MSC-transplanted mouse foot pads even after 15 min (Fig. 6B). Likewise, hematoxylin and eosin staining analysis revealed SG regeneration in 3D/PD-treated mice (Fig. 6C). GFP-positive cells were characterized as secretory lumen expressing K8, K18, and K19. Of note, the GFP-positive cells were highly distributed in K14-positive myoepithelial cells of SGs but were absent in K14-positive repaired epidermal wounds (Fig. 6, D and E). Thus, differentiated MSCs enabled directed restitution of damaged SG tissues both at the morphological and functional level.

(A) Schematic illustration of approaches for engineering iSGCs and transplantation. (B) Sweat test of mice treated with different cells. Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Histology of plantar region without treatment and transplantation of MSCs and iSGCs (scale bars, 200 m). (D) Involvement of GFP-labeled iSGCs in directed regeneration of SG tissue in thermal-injured mouse model (K14, red; GFP, green; DAPI, blue; scale bar, 200 m). (E) SG-specific markers K14, K19, K8, and K18 detected in regenerated SG tissue (arrows). (K14, K19, K8, and K18, red; GFP, green; scale bars, 50 m).

A potential gap in MSC-based therapy still exists between current understandings of MSC performance in vivo in their microenvironment and their intractability outside of that microenvironment (24). To regulate MSCs differentiation into the right phenotype, an appropriate microenvironment should be created in a precisely controlled spatial and temporal manner (25). Recent advances in innovative technologies such as bioprinting have enabled the complexities in the matrix design and fabrication of regenerative microenvironments (26). Our findings demonstrated that directed differentiation of MSCs into SGs in a 3D-printed matrix both in vitro and in vivo was feasible. In contrast to conventional tissue-engineering strategies of SG regeneration, the present 3D-printing approach for SG regeneration with overall morphology and function offered a rapid and accurate approach that may represent a ready-to-use therapeutic tool.

Furthermore, bioprinting MSCs successfully repaired the damaged SG in vivo, suggesting that it can improve the regenerative potential of exogenous differentiated MSCs, thereby leading to translational applications. Notably, the GFP-labeled MSC-derived glandular cells were highly distributed in K14-positive myoepithelial cells of newly formed SGs but were absent in K14-positive repaired epidermal wounds. Compared with no black dots were observed on single MSC-transplanted mouse foot pads, the black dots (representing sweating function) can be observed throughout the entire examination period, and the number increased within 10 min on MSC-bioprinted mouse foot pads. Thus, differentiated MSCs by 3D bioprinting enabled exclusive restitution of damaged SG tissues morphologically and functionally.

Although several studies indicated that engineering 3D microenvironments enabled better control of stem cell fates and effective regeneration of functional tissues (2730), there were no studies concerning the establishment of 3D-bioprinted microenvironments that can preferentially induce MSCs differentiating into glandular cells with multiple tissue phenotypes and overall functional tissue. To find an optimal microenvironment for promoting MSC differentiation into specialized progeny, biochemical properties are considered as the first parameter to ensure SG specification. In this study, we used mouse PD as the main composition of a tissue-specific ECM. As expected, this 3D-printed PD+ microenvironment drove the MSC fate decision to enhance the SG phenotypic profile of the differentiated cells. By ECM differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator of 3D-printed matrix for SG specification. TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development. Thus, we identified CTHRC1 as a specific factor during SG development. To our knowledge, this is the first demonstration of CTHRC1 involvement in dictating MSC differentiation to SG, highlighting a potential therapeutic tool for SG injury.

The 3D-printed matrix also provided architectural guides for further SG morphogenesis. Our results clearly show that the 3D spatial dimensionality allows for better cell proliferation and aggregation and affect the characteristics of phenotypic marker expression. Notably, the importance of 3D structural cues on MSC differentiation was further proved by MET-EMT process during differentiation, where the influences did not depend on the presence of biochemical cues. To fully elucidate the underlying mechanisms, we first examined how 3D structure regulating stem cell fate choices. According to our data, Hmox1 is highly up-regulated in 3D construct, which were supposed to response to hypoxia, with a previously documented role in MSC differentiation (31, 32). It is suggested that 3D microenvironment induced rapid cell aggregation leading to hypoxia and then activated the expression of Hmox1.

Through regulation of the expression of Hmox1 and addition or of CTHRC1 in the matrix, we confirmed that each of them is critical for SG reprogramming, respectively. Thus, biochemical and structural cues of 3D-printed matrix synergistically creating a microenvironment could enhance the accuracy and efficiency of MSC differentiation, thereby leading to resulting SG formation. Although we further need a more extensive study examining the role of other multiple cues and their possible overlap function in regulating MSC differentiation, our findings suggest that CTHRC1 and Hmox1 provide important signals that cooperatively modulate MSC lineage specification toward sweat glandular lineage. The 3D structure combined with PD stimulated the GO functional item of branch morphogenesis and gland formation, which might be induce by up-regulation of Bmp2 based on the verification of qPCR results. Although our results could not rule out the involvement of other factors and their possible overlapping role in regulating MSC lineage specification toward SGs, our findings together with several literatures suggested that BMP2 plays a critical role in inducing branch morphogenesis and gland formation (3335).

In summary, our findings represented a novel strategy of directing MSC differentiation for functional SG regeneration by using 3D bioprinting and pave the way for a potential therapeutic tool for other complex glandular tissues as well as further investigation into directed differentiation in 3D conditions. Specifically, we showed that biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation, and our results highlighted the importance of 3D-printed matrix cues as regulators of MSC fate decisions. This avenue opens up the intriguing possibility of shifting from genetic to microenvironmental manipulations of cell fate, which would be of particular interest for clinical applications of MSC-based therapies.

The main aim and design of the study was first to determine whether by using 3D-printed microenvironments, MSCs can be directed to differentiate and regenerate SGs both morphologically and functionally. Then, to investigate the underlying molecular mechanism of biochemical and structural cues of 3D-printed matrix involved in MSCs reprogramming. The primary aims of the study design were as follows: (i) cell aggregation and proliferation in a 3D-bioprinted construct; (ii) differentiation of MSCs at the cellular phenotype and functional levels in the 3D-bioprinted construct; (iii) the MET-EMT process during differentiation; (iv) differential protein expression of the SG niche in mice; (v) differential genes expression of MSCs in 3D-bioprinted construct; (vi) the key role of CTHRC1 and HMOX1 in MSCs reprogramming to SGCs; and (vii) functional properties of regenerated SG in vivo.

Gelatin (Sigma-Aldrich, USA) and sodium alginate (Sigma-Aldrich, USA) were dissolved in phosphate-buffered saline (PBS) at 15 and 1% (w/v), respectively. Both solutions were sterilized under 70C for 30 min three times at an interval of 30 min. The sterilized solutions were packed into 50-ml centrifuge tubes, stored at 4C, and incubated at 37C before use.

From wild-type C57/B16 mice (Huafukang Co., Beijing) aged 5 days old, dermal homogenates were prepared by homogenizing freshly collected hairless mouse PD with isotonic phosphate buffer (pH 7.4) for 20 min in an ice bath to obtain 25% (w/v) tissue suspension. The supernatant was obtained after centrifugation at 4C for 20 min at 10,000g. The DNA content was determined using Hoechst 33258 assay (Beyotime, Beijing). The fluorescence intensity was measured to assess the amount of remaining DNA within the decellularized ECMs and the native tissue using a fluorescence spectrophotometer (Thermo Scientific, Evolution 260 Bio, USA). The GAGs content was estimated via 1,9-dimethylmethylene blue solution staining. The absorbance was measured with microplate reader at wavelength of 492 nm. The standard curve was made using chondroitin sulfate A. The total COL (Collagen) content was determined via hydroxyproline assay. The absorbance of the samples was measured at 550 nm and quantified by referring to a standard curve made with hydroxyproline.

MSCs were bioprinted with matrix materials by using an extrusion-based 3D bioprinter (Regenovo Co., Bio-Architect PRO, Hangzhou). Briefly, 10 ml of gelatin solution (10% w/v) and 5 ml of alginate solution (2% w/v) were warmed under 37C for 20 min, gently mixed as bioink and used within 30 min. MSCs were collected from 100-mm dishes, dispersed into single cells, and 200 l of cell suspension was gently mixed with matrix material under room temperature with cell density 1 million ml1. PD (58 g/ml) was then gently mixed with bioink. Petri dishes at 60 mm were used as collecting plates in the 3D bioprinting process. Within a temperature-controlled chamber of the bioprinter, with temperature set within the gelation region of gelatin, the mixture of MSCs and matrix materials was bioprinted into a cylindrical construct layer by layer. The nozzle-insulation temperature and printing chamber temperature were set at 18 and 10C, respectively; nozzles with an inner diameter of 260 m were chosen for printing. The diameter of the cylindrical construct was 30 mm, with six layers in height. After the temperature-controlled bioprinting process, the printed 3D constructs were immersed in 100-mM calcium chloride (Sigma-Aldrich, USA) for 3 min for cross-linking, then washed with Dulbeccos modified Eagle medium (DMEM) (Gibco, USA) medium for three times. The whole printing process was finished in 10 min. The 3D cross-linked construct was cultured in DMEM in an atmosphere of 5% CO2 at 37C. The culture medium was changed to SG medium [contains 50% DMEM (Gibco, New York, NY) and 50% F12 (Gibco) supplemented with 5% fetal calf serum (Gibco), 1 ml/100 ml penicillin-streptomycin solution, 2 ng/ml liothyronine sodium (Gibco), 0.4 g/ml hydrocortisone succinate (Gibco), 10 ng/ml epidermal growth factor (PeproTech, Rocky Hill, NJ), and 1 ml/100 ml insulin-transferrin-selenium (Gibco)] 2 days later. The cell morphology was examined and recorded under an optical microscope (Olympus, CX40, Japan).

Fluorescent live/dead staining was used to determine cell viability in the 3D cell-loaded constructs according to the manufacturers instructions (Sigma-Aldrich, USA). Briefly, samples were gently washed in PBS three times. An amount of 1 M calcein acetoxymethyl (calcein AM) ester (Sigma-Aldrich, USA) and 2 M propidium iodide (Sigma-Aldrich, USA) was used to stain live cells (green) and dead cells (red) for 15 min while avoiding light. A laser scanning confocal microscopy system (Leica, TCSSP8, Germany) was used for image acquisition.

The cell-printed structure was harvested and fixed with a solution of 4% paraformaldehyde. The structure was embedded in optimal cutting temperature (OCT) compound (Sigma-Aldrich, USA) and sectioned 10-mm thick by using a cryotome (Leica, CM1950, Germany). The sliced samples were washed repeatedly with PBS solution to remove OCT compound and then permeabilized with a solution of 0.1% Triton X-100 (Sigma-Aldrich, USA) in PBS for 5 min. To reduce nonspecific background, sections were treated with 0.2% bovine serum albumin (Sigma-Aldrich, USA) solution in PBS for 20 min. To visualize iSGCs, sections were incubated with primary antibody overnight at 4C for anti-K8 (1:300), anti-K14 (1:300), anti-K18 (1:300), anti-K19 (1:300), anti-ATP1a1 (1:300), anti-Ki67 (1:300), antiN-cadherin (1:300), antiE-cadherin (1:300), anti-CTHRC1 (1:300), or anti-TSP1 (1:300; all Abcam, UK) and then incubated with secondary antibody for 2 hours at room temperature: Alexa Fluor 594 goat anti-rabbit (1:300), fluorescein isothiocyanate (FITC) goat anti-rabbit (1:300), FITC goat anti-mouse (1:300), or Alexa Fluor 594 goat anti-mouse (1:300; all Invitrogen, CA). Sections were also stained with 4,6-diamidino-2-phenylindole (Beyotime, Beijing) for 15 min. Stained samples were visualized, and images were captured under a confocal microscope.

To harvest the cells in the construct, the 3D constructs were dissolved by adding 55 mM sodium citrate and 20 mM EDTA (Sigma-Aldrich, USA) in 150 mM sodium chloride (Sigma-Aldrich, USA) for 5 min while gently shaking the petri dish for better dissolving. After transfer to 15-ml centrifuge tubes, the cell suspensions were centrifuged at 200 rpm for 3 min, and the supernatant liquid was removed to harvest cells for further analysis.

Total RNA was isolated from cells by using TRIzol reagent (Invitrogen, USA) following the manufacturers protocol. RNA concentration was measured by using a NanoPhotometer (Implen GmbH, P-330-31, Germany). Reverse transcription involved use of a complementary DNA synthesis kit (Takara, China). Gene expression was analyzed quantitatively by using SYBR green with the 7500 Real-Time PCR System (Takara, China). The primers and probes for genes were designed on the basis of published gene sequences (table S1) (National Center for Biotechnology Information and PubMed). The expression of each gene was normalized to that for glyceraldehyde-3-phosphate dehydrogenase and analyzed by the 2-CT method. Each sample was assessed in triplicate.

The culture medium was changed to SG medium with 2 mM CaCl2 for at least 24 hours, and cells were loaded with fluo-3/AM (Invitrogen, CA) at a final concentration of 5 M for 30 min at room temperature. After three washes with calcium-free PBS, 10 M acetylcholine (Sigma-Aldrich, USA) was added to cells. The change in the Fluo 3 fluorescent signal was recorded under a laser scanning confocal microscopy.

Cell proliferation was evaluated through CCK-8 (Cell counting kit-8) assay. Briefly, cells were seeded in 96-well plates at the appropriate concentration and cultured at 37C in an incubator for 4 hours. When cells were adhered, 10 l of CCK-8 working buffer was added into the 96-well plates and incubated at 37C for 1 hour. Absorbance at 450 nm was measured with a microplate reader (Tecan, SPARK 10M, Austria).

Proteomics of mouse PD and DD involved use of isobaric tags for relative and absolute quantification (iTRAQ) in BGI Company, with differentially expressed proteins detected in PD versus DD. Twofold greater difference in expression was considered significant for further study.

Tissues were grinded and lysed in radioimmunoprecipitation assay buffer (Beyotime, Nanjing). Proteins were separated by 12% SDSpolyacrylamide gel electrophoresis and transferred to a methanol-activated polyvinylidene difluoride membrane (GE Healthcare, USA). The membrane was blocked for 1 hour in PBS with Tween 20 containing 5% bovine serum albumin (Sigma-Aldrich, USA) and probed with the antibodies anti-CTHRC1 (1:1000) and anti-TSP1 (1:1000; both Abcam, UK) overnight at 4C. After 2 hours of incubation with goat anti-rabbit horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, CA), the protein bands were detected by using luminal reagent (GE Healthcare, ImageQuant LAS 4000, USA).

Total RNA was prepared with TRIzol (Invitrogen), and RNA sequencing was performed using HiSeq 2500 (Illumina). Genes with false discovery rate < 0.05, fold difference > 2.0, and mean log intensity > 2.0 were considered to be significant.

CAPE or Snpp was gently mixed with bioink at a concentration of 10 M. Physiological concentration of CTHRC1 was measured by enzyme linked immunosorbent assay (ELISA) (80 ng/ml), and then recombinant CTHRC1 or CTHRC1 antibody was added into the bioink at a concentration of 0.4 g/ml. The effect of inhibitor and activator was estimated by qRT-PCR or ELISA.

Mice were anesthetized with pentobarbital (100 mg/kg) and received subcutaneous buprenorphine (0.1 mg/kg) preoperatively. Full-thickness scald injuries were created on paw pads with soldering station (Weller, WSD81, Germany). Mice recovered in clean cages with paper bedding to prevent irritation or infection. Mice were monitored daily and euthanized at 30 days after wounding. Mice were maintained in an Association for Assessment and Accreditation of Laboratory Animal Careaccredited animal facility, and procedures were performed with Institutional Animal Care and Use Committeeapproved protocols.

MSCs in 3D-printed constructs with PD were cultured with DMEM for 2 days and then replaced with SG medium. The SG medium was changed every 2 days, and cells were harvested on day 12. The K18+ iSGCs were sorting through flow cytometry and injected into the paw pads (1 106 cells/50 l) of the mouse burn model by using Microliter syringes (Hamilton, 7655-01, USA). Then, mice were euthanized after 14 days; feet were excised and fixed with 10% formalin (Sigma-Aldrich, USA) overnight for paraffin sections and immunohistological analysis.

The foot pads of anesthetized treated mice were first painted with 2% (w/v) iodine/ethanol solution then with starch/castor oil solution (1 g/ml) (Sigma-Aldrich, USA). After drying, 50 l of 100 M acetylcholine (Sigma-Aldrich, USA) was injected subcutaneously into paws of mice. Pictures of the mouse foot pads were taken after 5, 10, and 15 min.

All data were presented as means SEM. Statistical analyses were performed using GraphPad Prism7 statistical software (GraphPad, USA). Significant differences were calculated by analysis of variance (ANOVA), followed by the Bonferroni test when performing multiple comparisons between groups. P < 0.05 was considered as a statistically significant difference.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1094/DC1

Fig. S1. Biocompatibility of 3D-bioprinted construct and cellular morphology in 2D monolayer culture.

Fig. S2. Expression of SG-specific and secretion-related markers in MSCs and SG cells in vitro.

Fig. S3. Transcriptional and translational expression of epithelial and mesenchymal markers in 3D-bioprinted cells with and without PD.

Fig. S4. Expression of N- and E-cadherin in MSCs and SG cells in 2D monolayer culture.

Fig. S5. Proteomic microarray assay of differential gene expression between PD and DD ECM in postnatal mice.

Fig. S6. GO term analysis of differentially expressed pathways.

Fig. S7. Heat maps illustrating differential expression of genes implicated in ECM organization, cell division, and gland and branch morphogenesis.

Fig. S8. The expression of Hmox1 and the concentration of CTHRC1 on treatment and the related effects on cell proliferation.

Fig. S9. The expression of K8 and K18 with Hmox1 and CTHRC1 regulation.

Table S1. Primers for qRT-PCR of all the genes.

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 study was supported in part by the National Nature Science Foundation of China (81571909, 81701906, 81830064, and 81721092), the National Key Research Development Plan (2017YFC1103300), Military Logistics Research Key Project (AWS17J005), and Fostering Funds of Chinese PLA General Hospital for National Distinguished Young Scholar Science Fund (2017-JQPY-002). Author contributions: B.Y. and S.H. were responsible for the design and primary technical process, conducted the experiments, collected and analyzed data, and wrote the manuscript. Y.W. and R.W. helped perform the main experiments. Y.Z. and T.H. participated in the 3D printing. W.S. and Z.L. participated in cell experiments and postexamination. S.H. and X.F. collectively oversaw the collection of data and data interpretation and revised 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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Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration - Science...

FORMS of blood cancer, such as leukaemia or lymphoma, are the fifth most common cancer, and the third biggest cause of cancer deaths.

Yet warning signs can be so unlike those of other cancers, that its often diagnosed at a very late stage.

Research by UK blood cancer charity Bloodwise (bloodwise.org.uk) found more than a third of sufferers had to visit their GP three or more times with symptoms before being a hospital referral. This makes it the worst performing cancer in terms of early diagnosis.

Why is it so difficult to spot? Blood cancers, which stop blood stem cells from working normally and can make you weak and prone to infections, have three main types with many different variations. These variations have numerous diverse symptoms, which can often be mistaken for other less serious conditions.

Not all signs of blood cancer are easily identifiable, or are associated with typical symptoms of cancer, such as a lump or abnormal mole, says haematologist Dr Manos Nikolousis, a medical consultant with UK blood cancer charity DKMS.

Blood cancer often presents in ways which are most commonly associated with unrelated and less serious illnesses, like a cold or flu. In other circumstances, patients notice a change in their body which they cant quite put their finger on.

One of the treatments for blood cancer is a stem cell transplant that restores blood-forming stem cells in patients whove had theirs destroyed by very high doses of chemotherapy and/or radiotherapy. But Nikolousis points out that only one in three blood cancer patients who need a transplant find a matching blood stem cell donor in their family. The remaining two-thirds have to rely on an unrelated donor, which significantly reduces their chance of finding a crucial match.

Here, Nikolousis outlines some blood cancer symptoms and warning signs...

Musculoskeletal pain in muscles, joints, tendons, bones or structures that support the limbs, neck or back.

One of the most common symptoms associated with blood cancer. The frequency and lasting impact of bruising can be a key warning sign, so its important to book an appointment with your GP if this develops.

Unexplained and persistent tiredness is one of the biggest tell-tale signs of blood cancer. People who have cancer-related fatigue find it incredibly challenging to complete simple tasks that we tend to take for granted.

The lymph nodes are small lumps of tissue that contain white blood cells. When inflamed, they can be felt as lumps under the skin; most commonly in the neck, armpit or groin area.

There may be new headaches that feel different. Theyre likely to occur frequently and be severe and long-lasting.

Persistent abdominal discomfort, presenting as a sharp pain or a sense of feeling full.

This can be described as a feeling of pins and needles/numbness that moves up to the legs, or from fingers to the arms.

This can feel like a fluttering, a sudden thump or a fast pounding sensation in the chest. It can also be felt in the neck or ear when lying down.

People may describe this as feeling mentally drained or dizzy.

Blood cancer patients may have continuous trouble falling asleep or staying asleep.

Persistent and irritable, this may be experienced all over the body, or in isolated spots.

These symptoms are common and dont automatically mean you have cancer. But if you notice any unusual or ongoing changes, its always best to see your doctor and get checked.

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Eleven symptoms of blood cancer that everybody needs to know about... - Echo Live

Buyers looking to own a private, well-stocked fishing pond or enjoy the simplicity of a quiet paddle across 18 acres of tranquil water will find all of that and a 5,619-square-foot custom contemporary home at 355 Pond St. in Uxbridge.

Set back from the road on more than 56 scenic acres, the 10-room home, on the market for $1.1 million, offers unique upgrades such as cedar paneled ceilings and 14-karate-gold plated fixtures in the master bath.

The house also features a 2,400-square-foot, wraparound deck that overlooks Lee Pond.

Owner Dr. Charles A. Vacanti said his favorite feature of the property is its pond, which is stocked upstream along Emerson Brook by two fishing clubs.

Both of their ponds drain into our 18-acre pond, he said. It may be the best fishing hole in New England.

Listing agent Gary Smith of Mendon Area Real Estate called the pond and its surrounding property beautiful, while his favorite feature in the home is the spectacular family room.

The sunken combination family and dining room share a wood burning stove with a two-story stone surround. The cathedral ceilings are paneled in cedar and hold skylights and fans. Light from two panels of floor to ceiling glass brightens this room that gives access to the deck.

The modern, eat-in kitchen holds a center island, custom-built cabinetry, granite countertops and large pantry.

The upstairs hallway is an open loft overlooking the family/dining room.

The master suite offers scenic views through a wall of glass with a door that opens to a balcony. The master bath has marble flooring, a cathedral ceiling with skylights, custom built closet and cabinets, a spa hot tub, and large stall shower.

Also upstairs are an office and gym space.

Dr. Vacanti and his wife, Linda, bought the property in 1998.

Since then, the couple has replaced all 27 skylights, all external doors and windows, and the roof. The installation of solar panels, Dr. Vacanti said, has reduced annual utility costs to nearly zero.

Dr. Vacanti is widely known for his research work in stem cell and tissue engineering. While at the University of Massachusetts Medical School in 1996, he grew new cartilage in the shape of a human ear under the skin of a lab mouse. The Vacanti Mouse was part of research on organ generation and the first demonstration of growing new tissue from cells in a lab.

As for the property, Smith called it a beautiful high and dry pasture that just begs for equestrian use, a corporate retreat, Bed and Breakfast, or Rod and Gun Club.

The property is minutes from Route 146.

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House of the Week: Uxbridge property is a sportsmans paradise - Worcester Telegram

New Delhi: The usage of stem cells to cure or treat a disease or repair the injured tissue is defined as stem cell therapy. The best example of the stem cell treatment is seen in patients suffering from restoring the vision of the damaged eyes, grafting of the skin in severe burnt conditions.

Stem cell treatments for brain or neural diseases like Parkinsons and Alzheimers disease, multiple sclerosis, preventing heart strokes, curing diabetes, kidney disorders, autism, and spinal cord injuries are progressively making their way.

Undifferentiated cells that are able to differentiate and transform into any type of cells of the body when and where needed. They have an enormous potential to repair, heal and regenerate. Stem cells come from blood, bone marrow, umbilical cord blood and adipose tissue.

Autologous stem cell therapy: Patient receives stem cells from his/her own body

Allogeneic stem cell therapy: Patient receives the stem cells donated by another individual

Autologous stem cell therapy is better than allogeneic stem cell therapy as chances of mismatching are not there and they pose the minimum risk of immune rejection. Also, no side effects or adverse effects are seen as a persons own blood cells are used. They start the healing process immediately in a natural way.

The usage of stem cells to cure or treat a disease or repair the injured tissue is defined as stem cell therapy. Stem cells can be obtained from the bone marrow, adipose tissues etc. Due to their tremendous potential to prevent and to treat various health conditions and to repair the injured tissues global research investigation is continuously being done as to explore the maximum advantage of these cell lines.

The best example of the stem cell treatment is seen in patients suffering from restoring the vision of the damaged eyes, grafting of the skin in severe burnt conditions. Stem cell treatments for brain or neural diseases like Parkinsons and Alzheimers disease, multiple sclerosis, preventing heart strokes, curing diabetes, kidney disorders, autism, and spinal cord injuries are progressively making their way.

Depending upon the disease, different stem cell source can be used in a specific condition. The procedure may involve the extraction of stem cells from adipose tissue-derived stem cells with the combination of PRP (Platelet-rich plasma) or can be obtained from bone marrow that can differentiate into progenitor cells that differentiate into various other tissues which can help in the therapy.

The stem cells are isolated from the bone marrow or adipose tissues followed by their processing and enrichment under sterile conditions. These activated stem cells are placed back into the patients body at the target site for repairing the damaged tissue. It is necessary that the stem cells are injected in the specific area of injury as only then the desired results will be achieved.

Adipose stem cells are preferred over bone marrow stem cells as they are easy to isolate and contain a higher number of stem cells.

The stem cells injections are gaining much interest because it is devoid of the painful procedure, takes less time in comparison to surgery, there are no host and recipient rejections as stem cells are harvested from the patients body itself and a targeted delivery system is available.

The stem cells obtained are processed in a sophisticated stem cell lab and after activation is inserted back into the host with the help of intravenous, intramuscular, intraarterial, intradermal and intrathecal injections as per the requirement of the treatment process.

What is the use of anaesthetics and why? Usually, local anaesthetics are used during a stem cell procedure to numb the area but sometimes general anaesthesia is also given while extracting the stem cells from bone marrow. But it is necessary to find out what anaesthetic your doctor uses during orthopaedic stem cell treatments.

A number of anaesthetics have been found to kill the stem cells thus; the treatments end result will greatly depend on the use of anaesthetics. Some anaesthetics very well sync with the stem cell and hence, aid in the treatment.

Stem cells are to be extracted and processed in a clean room, under aseptic conditions maintaining a controlled environment. The doctor should explain the entire process and the number of viable stem cells infused into the patient during the process. Also, the precision of the injections to provide good quality of stem cells at the site of injury will help in better and faster recovery of the patients damaged area.

Cost of the treatment and its duration varies from one patient to another. The disease which needs to be cured, the severity, age factor, health condition, etc, define the duration of the therapy. One may respond during the treatment phase itself while the other may show results after a few sessions or weeks. Depending upon the disease diagnosed, the stem cells extracted, duration of the therapy, other adjuvants used in the process, the cost of the stem cell therapy can vary.

It is essential that after the stem cell therapy the patient should visit the stem cell doctor for recuperation therapies. The primary goals of such therapy is the prevention of secondary complications, analysis of the recovery of motor, sensory and all the bodily functioning, psychological support/counselling for depression, mood swings or anxiety etc. and reintegration into the community.

There can be different sets of precautions which need to be followed at various steps for the recovery of the damaged tissues. The treatment and post-treatment conditions may vary from person to person depending upon the disease and the severity.

Stem cell therapy has shown results in treating serious ailments like leukaemia, grafting tissues, autism, orthopaedic conditions and skin problems etc. Stem Cell Therapy has been successfully used in the treatment of around 80 serious disorders.

Survival rates among patients who received stem cell treatment are significantly high, whether cell donors are related or unrelated to them. With the ongoing research around the world, scientists are exploring new possibilities in which a number of life-threatening diseases can be prevented and cured hence, the stem cells have proved to be promising in the near future as many aspects are yet to be revealed.

Read: Egg stem cells do not exist, says researchers

-IANS

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10 things to know about stem cell therapy - Newsd.in

Technology Networksrecently had the pleasureof speaking with Bruce Steel, CEOEquillium, Inc. to learn more about how they are leveraging their comprehensive understanding of immunobiology to develop novel treatments for inflammatory and autoimmune disorders.Bruce provides insight on the various indications the company is currently developing treatments for, elaborates on the clinical progress of their initial product candidate EQ001 (itolizumab), and explains the role CD6 plays in autoimmunity and how it can be targeted therapeutically.

Laura Lansdowne (LL): How can immunobiology be harnessed to develop therapeutics?Bruce Steel (BS): The role of the immune system is to defend the body against foreign organisms and cells, including cancerous cells, and in doing so, it must distinguish accurately between self- and non-self-entities a process called tolerance. Autoimmunity is an immune response directed against the bodys own healthy cells and tissues and is the underlying process in many inflammatory diseases. Autoimmunity results from a loss of tolerance caused in part by an imbalance in the relationship between effector T cells and regulatory T cells. Therefore, developing therapeutics, such as EQ001 (itolizumab), that target these critical regulators of immune activation pathways has the potential dramatically improve the lives of patients with severe autoimmune and inflammatory disorders.LL: What indications are you currently developing treatments for?BS: We select target indications based on three primary criteria: strong scientific rationale for why itolizumab has potential to be a best-in-class therapeutic approach, areas of high unmet medical need where there are little or no treatments available today, and indications where we believe there is an attractive future commercial opportunity. Today we have ongoing clinical trials with itolizumab in acute graft-versus-host disease (GVHD), uncontrolled asthma and lupus/lupus nephritis.

While these three indications are our initial areas of focus, we believe itolizumab has the potential to be developed in other areas such as transplant science, systemic autoimmunity, pulmonary, neurologic, gastrointestinal, renal, vascular, ophthalmic and dermatologic disorders.We licensed itolizumab from our partner Biocon Limited who developed the drug and received regulatory approval in India for the treatment of psoriasis.LL: Can you tell us more about the mechanism of your drug candidate EQ001 (itolizumab)?BS: Itolizumab is a clinical-stage, first-in-class monoclonal antibody that selectively targets the CD6-ALCAM pathway, which plays a central role in modulating the activity and trafficking of effector T cells that drive a number of immune-inflammatory diseases.LL: What role does CD6 play in autoimmunity?BS: CD6 is a novel co-stimulatory receptor that uniquely modulates T cell activity and trafficking. It is a key checkpoint in regulating effector T cells that are central to autoimmune responses. CD6 binds activated leukocyte cell adhesion molecule (ALCAM). ALCAM is expressed on both antigen-presenting cells and tissue including the skin, gut, lung and kidney.In preclinical studies, blockade of CD6 with itolizumab leads to reduction in effector T cell activation and proliferation. Additionally, inhibiting the binding of ALCAM to CD6 with itolizumab modulates lymphocyte trafficking and reduces effector T cell infiltration into inflamed tissues.

Our work with CD6 builds upon the research conducted from researchers at the Dana-Farber Cancer Institute, our partner Biocon, and other leading academic centers. Today there are numerous peer reviewed publications related to the novel CD6 checkpoint receptor and targeting the CD6-ALCAM pathway.LL: Can you elaborate on the clinical progress of itolizumab?BS: Itolizumab is currently being studied in three different indications: acute graft-versus-host disease (aGVHD), uncontrolled moderate to severe asthma and lupus/lupus nephritis. This is an important catalyst year for Equillium as we expect initial data from all three programs in 2H 2020.

Each of these studies will allow us to understand the safety of itolizumab in these different disease areas and understand what potential dose we will carry forward. This is important as we consider making a larger investment in Phase II studies to advance the program.Bruce Steel was speaking with Laura Elizabeth Lansdowne, Senior Science Writer for Technology Networks.

Excerpt from:
Exploiting Immunobiology To Treat Severe Autoimmune and Inflammatory Disorders - Technology Networks

Avoid close contact with sick patients. Stay home if youre feeling unwell. Scrub your hands with soap and water for at least 20 seconds and for goodness sake, stop touching your face.

By now, youve probably heard or seen the advice from the Centers for Disease Control and Prevention (CDC) for staving off COVID-19, the viral epidemic ricocheting across the globe. Most cases of the disease are mild, triggering cold-like symptoms including fever, fatigue, dry cough and shortness of breath. The death rate appears to be lowabout two or three percent, perhaps much less. But the virus responsible, called SARS-CoV-2, is a fearsomely fast spreader, hopping from person to person through the droplets produced by sneezes and coughs. Since COVID-19 was first detected in Chinas Hubei province in December 2019, nearly 100,000 confirmed cases have been reported worldwide, with many more to come.

To curb the virus spread, experts stress the importance of hand hygiene: keeping your hands clean by regularly lathering up with soap and water, or, as a solid second choice, thoroughly rubbing them down with an alcohol-based sanitizer. That might sound like simple, even inconsequential advice. But such commonplace practices can be surprisingly powerful weapons in the war against infectious disease.

[Washing your hands] is one of the most important ways to interrupt transmission of viruses or other pathogens, says Sallie Permar, a physician and infectious disease researcher at Duke University. It can have a major impact on an outbreak.

In the strictest sense of the word, viruses arent technically alive. Unlike most other microbes, which can grow and reproduce on their own, viruses must invade a host such as a human cell to manufacture more of themselves. Without a living organism to hijack, viruses cant cause illness. Yet viral particles are hardy enough to remain active for a while outside of the host, with some staying infectious for hours, days or weeks. For this reason, viruses can easily spread unnoticed, especially when infected individuals dont always exhibit symptomsas appears to be the case with COVID-19.

Researchers are still nailing down the details of exactly how SARS-CoV-2 is transmitted and how resilient it is outside the body. Because the virus seems to hang out in mucus and other airway fluids, it almost certainly spreads when infected individuals cough or sneeze. Released into the air, infectious droplets can land on another person or a frequently touched surface like a doorknob, shopping cart or subway seat. The virus can also transfer through handshakes after someone carrying the virus sneezes or coughs into their hand.

After that, its a short trip for the virus from hand to head. Researchers estimate that, on average, humans touch their faces upwards of 20 times an hour, with about 44 percent of these encounters involving eyes, mouths and nosessome of the quickest entry points into the bodys interior.

Breaking this chain of transmission can help stem the spread of disease, says Chidiebere Akusobi, an infectious disease researcher at Harvards School of Public Health. Sneezing or coughing into your elbow can keep mucus off your mitts; noticing when your hand drifts towards your face can help you reduce the habit.

All this public-health-minded advice boils down to a game of keep away. To actually infect a person, viruses must first get inside the body, where they can infect living cellsso if one lands on your hands, the best next move is to remove or destroy it.

The most important step to curbing infection may be hand-washing, especially before eating food, after using the bathroom and after caring for someone with symptoms. Its simply the best method to limit transmission, says Kellie Jurado, a virologist at the University of Pennsylvanias Perelman School of Medicine. You can prevent yourself from being infected as well as transmitting to others.

According to the CDC, you should wet your handsfront and backwith clean, running water; lather up with soap, paying mind to the easily-forgotten spaces between your fingers and beneath your nails; scrub for at least 20 seconds; then rinse and dry. (Pro tip: If counting bores you or youre sick of the birthday song, try the chorus of these popular songs to keep track.)

Done properly, this process accomplishes several virus-taming tasks. First, the potent trifecta of lathering, scrubbing and rinsing physically removes pathogens from your skin, says Shirlee Wohl, a virologist and epidemiologist at Johns Hopkins University.

In many ways, soap molecules are ideal for the task at hand. Soap can incapacitate SARS-CoV-2 and other viruses that have an outer coating called an envelope, which helps the pathogens latch onto and invade new cells. Viral envelopes and soap molecules both contain fatty substances that tend to interact with each other when placed in close proximity, breaking up the envelopes and incapacitating the pathogen. Basically, the viruses become unable to infect a human cell, Permar says.

Alcohol-based hand sanitizers also target these vulnerable viral envelopes, but in a slightly different way. While soap physically dismantles the envelope using brute force, alcohol changes the envelopes chemical properties, making it less stable and more permeable to the outside world, says Benhur Lee, a microbiologist at the Icahn School of Medicine at Mount Sinai. (Note that alcohol here means a chemical like ethanol or isopropyl alcoholnot a beverage like vodka, which contains only some ethanol.)

Alcohol also can penetrate deep into the pathogens interior, wreaking havoc on proteins throughout the virus. (Importantly, not all viruses come with outer envelopes. Those that dont, like the viruses that cause HPV and polio, wont be susceptible to soap, and to some extent alcohol, in the same way.)

Hand sanitizers made without alcohollike some marketed as baby-safe or naturalwont have the same effect. The CDC recommends searching for a product with at least 60 percent alcohol contentthe minimum concentration found to be effective in past studies. (Some water is necessary to unravel the pathogens proteins, so 100 percent alcohol isnt a good option.)

As with hand-washing, timing matters with sanitizers. After squirting a dollop onto your palm, rub it all over your hands, front and back, until theyre completely drywithout wiping them off on a towel, which could keep the sanitizer from finishing its job, Jurado says.,

But hand sanitizers come with drawbacks. For most people, using these products is less intuitive than hand-washing, and the CDC notes that many people dont follow the instructions for proper application. Hand sanitizers also dont jettison microbes off skin like soap, which is formulated to lift oily schmutz off surfaces, Akusobi says.

Soap emulsifies things like dirt really well, he says. When you have a dirty plate, you dont want to use alcoholthat would help sterilize it, but not clean it.

Similarly, anytime the grit is visible on your hands, dont grab the hand sanitizer; only a full 20 seconds (or more) of scrubbing with soapy water will do. All told, hand sanitizer should not be considered a replacement for soap and water, Lee says. If I have access to soap and water, I will use it.

Technically, it is possible to overdo it with both hand-washing and hand sanitizing, Akusobi says. If your skin is chronically dry and cracking, thats no good. You could be exposing yourself to other infections, he says. But it would take a lot to get to that point.

In recent weeks, hand sanitizers have been flying off the shelves, leading to shortages and even prompting some retailers to ration their supplies. Some people have begun brewing up hand sanitizers at home based on online recipes.

Many caution against this DIY approach, as the end products cant be quality controlled for effectiveness, uniformity or safety, says Eric Rubin, an infectious disease researcher at Harvards School of Public Health. On average, one would imagine that [a homemade sanitizer] would not work as well, so it would be a mistake to rely on it, he says.

As more information on SARS-CoV-2 and COVID-19 emerges, experts stress the importance of awareness. Even as the news changes and evolves, peoples vigilance shouldnt.

Do the small things you need to do to physically and mentally prepare for whats next, Wohl says. But dont panic. That never helps anybody.

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Why Is Washing Your Hands So Important Anyway? - Smithsonian

Recent developments in the field of stem cell research are paving a path towards a radical shift in the way we diagnose and treat dementia. Stem cells have excited scientists for years and research groups across the globe are using them to advance modern medicine. Using stem cells to aid the fight against dementia is perhaps one of the most critical applications of the technology. Dementia is the leading cause of death in the UK, sixth in US and fifth globally, with an estimated 50 million people currently affected.

The term dementia does not relate to a single disease, but more an array of symptoms that can arise from multiple conditions. The most common is Alzheimers disease (AD) which accounts for up to 80% of all cases. Dementia itself is caused by the death of cells that make up the complex circuitry of our brains and an eventual loss of large portions of the brain. Patients suffering with dementia often exhibit the same general symptoms such as confusion, memory loss and an inability to perform day to day functions. It is a debilitating condition that often strikes the most vulnerable members of society and, consequently, many research groups around the globe work to try to understand dementia-causing diseases to provide better diagnostic and treatment platforms.

In 2007, a research group at Kyoto University in Japan published a study with the potential to change the face of research into dementia along with many other fields. Professor Shinya Yamanaka and his research team developed a method whereby stem cells (cells that can be transformed/differentiated into cells from any tissue) could be generated from a sample of skin. The study, which resulted in a 2012 Nobel Prize for Prof. Yamanaka, demonstrated that skin cells could be isolated from a patient and genetically reprogrammed into induced pluripotent stem cells (iPSCs). In short, this technology made it possible to generate and study brain cells from a patient with dementia without having to remove any of their brain. All they would need to do is provide scientists with a sample of skin.

Since this development, research groups around the globe have started using iPSCs from many patients with dementia in order to understand the biological mechanisms that underlie disease. Dr Eric Hill runs a research group at Aston University in the UK that specializes in iPSCs for dementia research and he had the following to say about the technology:

Its really exciting because it allows us to study cells with genetic mutations that are patient specific. We can get a much better picture of what is actually happening in the brains of these patients. We can now generate all the different cell types found in the human brain and understand how they function together and map the changes that result in disease.

The latter was perhaps most powerfully demonstrated in a study published by a team at the University of North Carolina, led by Professor Hansang Cho. The team was able to generate three key cell subtypes that play important roles in brain function; study the impact of mutations associated with Alzheimers disease; and even replicate some of the core malfunctions found to trigger disease in the brains of patients.

Studies like this are of significance because a large part of the focus in dementia research is on trying to understand how such changes in function arise. When a patient is diagnosed with a disease such as Alzheimers it is often too late for effective treatment. Scientists, instead, seek to elucidate those early changes in brain cell function in order to diagnose patients earlier to give more time for treatment. It is very much a case of prevention being better than a cure. Dr Hill provided an encouraging statement regarding this:

When we generate brain cells from iPSCs the cells we get are developmentally very young. What is interesting is the fact we still see differences between cells from dementia patients versus healthy patients suggesting we could find markers to help us detect and prevent disease some years before it develops.

Despite such promise, however, iPSCs have yet to provide the field of dementia research with that big break. Multiple treatments have progressed into clinical trials since the technology first emerged but no therapies have been approved. Drugs that show promise in the lab fail to deliver on their potential in patient clinical trials, sending researchers back to square one.

We should not be disheartened by this, however, and should instead view it as space into which the technology of using iPSCs to study dementia can grow. A lot of drugs fail in clinical trials because the platforms used to run initial tests dont provide scientists with a wide enough perspective of how those drugs will influence human cells. Additionally, many preclinical studies use animals with dementia-causing disease artificially induced into them. Studies like this often fail to translate into humans because the initial data is not from a human perspective. This is where researchers like Dr. Hill think iPSCs can provide us with an advantage:

iPSCs could provide us with much better platforms for screening drugs to treat and prevent these diseases. They can really add to what we already have, and while we might not be able to grow a full human brain, we can generate the cells that provide the building blocks for one. They give us the chance to screen new therapies more efficiently, better test their effectiveness and reduce the amount of animal use in dementia research.

Dr Hill is not alone in seeing the promise of using iPSCs to find better treatments for preventing the progression of dementia. Multiple research groups around the world have shown the potential of iPSC-derived brain cells for studying the effectiveness of new therapies.

In the last 12 months we have observed a wave of new studies using iPSCs to try to develop better treatments for diseases like Alzheimers, Parkinsons, Huntingtons disease and ALS. From studies in the University of California identifying cholesterol metabolism as a potential target to treating Alzheimers to studies in Luxembourg helping us find better treatments for Parkinsons, it is easy to see why the global effort to get that big break from iPSCs continues to gain interest. We might still be waiting for that next Noble Prize-winning discovery that will improve the lives of millions of patients but the collective effort of iPSC research groups across the world brings us a step closer with every study they publish. Dementia may, one day, be a thing of the past and iPSC research will likely be a significant part in getting us there.

Sam Moxon has a PhD in regenerative medicine and is currently involved in dementia research. He is a freelance writer with an interest in the development of new technologies to diagnose and treat degenerative diseases. Follow him on Twitter @DrSamMoxon

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Searching for the 'big break' that could turn stem cells into a weapon against dementia - Genetic Literacy Project

When an animal, such as a dog or cat, is sick or unwell, its first natural instinct is to refuse food. Their body automatically cuts down appetite till they recover. They may drink water, but nothing else. This is bodys intelligence taking over to heal and recover.Intuitive eatingHumans arent any different. At least, intuitively. All of us are products of nature and we have been designed the same. When we call in sick, the first thing that happens is that our appetites shut down too. Our taste of mouth changes and our sensitivity to smell and taste is numbed. This is our bodys intelligence and subtle biofeedback trying to tell us that its time we give our body some rest.

This is fasting in a way and has been practised since the longest time across all religions. Fasting is inbuilt in us and is considered medicinal if you actually go back and read its history. It is our bodys uncomplicated way of cleaning and healing. Unfortunately, we have moved too far from nature and our natural instincts. We are constantly eating and munching even when sick, thereby compromising our own immunity and masking the inbuilt mechanism to heal.Take a break

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Dont feed sickness - Times of India

A class action lawsuit says that Peter Thomas Roth makes false anti-aging claims about two of its product lines the Rose Stem Gel products and the Water Drench line of products. Plaintiffs Angela Clair, Bonnie McDonald, and Miley-Isabella Oien say they purchased products from the two lines in question, believing the claims made by Peter Thomas Roth about the capability of the products.The Peter Thomas Roth class action claims that the skincare company falsely advertises that the line of Rose Stem Cell products can improve and repair human skin because of the presence of rose stem cells in them. Ring of Fires Farron Cousins discusses this with Scott Hardy, the President of Top Class Actions.

Transcript:

*This transcript was generated by a third-party transcription software company, so please excuse any typos.

Farron Cousins:Most people think when they buy a product that contains some kind of stem cell, that theyre getting a top of the line product that is definitely going to work. After all, we all know the benefits of stem cells. They have been involved in countless medical breakthroughs even up to this day. So yeah, consumers across the planet would love to buy a product containing stem cells, but unfortunately theres different kinds of stem cells and that has now gotten one company, Peter Thomas Roth, in quite a bit of trouble with a new lawsuit.

Joining me now to explain this is Scott Hardy with Top Class Actions and Scott, yeah, everybody wants to get their hands on products with stem cells, especially in this case, anti-aging creams because you assume you put that on your face with these stem cells, its obviously gonna do some really great things because stem cells can do that. But as I mentioned, different kinds of stem cells here. You know, this isnt the human stem cells. This is something else, isnt it?

Scott Hardy:Thats right. You have Peter Thomas Roth and their anti-aging products, which are promising, according to this glass action, some amazing results. And with these stem cells, they, you know, like you said, you hear stem cells, you say, oh, I could rub this in. Stem cells are magical. They will make me look younger. Itll be great. Well, in this they had their Rose Stem Gel products, the Peter Thomas Roth Rose Stem Gel products, which have stem cells from roses. Now, Im not quite sure how that helps us as humans, but according to Peter Thomas Roth and their marketing agreements, these stem cells can have anti-aging and restorative benefits. But as like a lot of these class actions, the class actions saying that theres no science behind it that supports that. They also have their Water Drench line and the Water Drench line says that it draws moisture from the atmosphere into the users skin.

I mean, that is special and claims that the product will hold a thousand times its weight in water and provide hydration up to 72 hours. But again, unfortunately according to this class action, theres just no science to back it up. So you have these folks that are buying these products, hoping that it will help clear away their wrinkles, restore their face. You know, any age marks theyll, thats going to help, thats going to help them disappear with these Rose stem cells and this amazing Peter Thomas Roth Water Drench. But the science isnt backing it up is what the plaintiff attorneys are saying.

Farron Cousins:Well, and that, you know, makes total sense because human stem cells work because theyre cells that are not necessarily programmed for a specific function. So when you put them in the body, when you put them in a certain part of the body, they will kind of take on the role that the other cells in that area do. For example, you have a damaged liver and you get injected with stem cells in the liver, they will become or can become, excuse me, liver cells. So you would think if youre putting stem cells on your face, theyre going to become human skin cells. But you cant do that with a different, I mean, were not even talking about a different animal here. Were talking about a different organism all together. Roses, Im sorry, but human cells cannot bond with plant cells like that.

I mean, that, thats not a thing that is even scientifically possible. Maybe somewhere in the future were going to be able to merge with plants, I doubt it, but this company is saying, no, no, no. A stem cell is a stem cell, right? This is going to do well for you. And then I got to say with this Water Drench product, this would be a miracle product all around the world. You know, help your body draw in moisture from just the air. Oh my God, think of the problems we could solve. This, this, this truly is snake oil in my opinion, reading this, looking at these complaints, this stuff is pure snake oil, man.

Scott Hardy:Yeah, I mean thats what were seeing here. Were getting a bunch of comments on this. People that have bought these products and didnt see any results, and thats the big problem is that if youre going to promise these results, if youre going to say that we have this special benefit, we have these special ingredients that make our product amazing, then it needs to deliver. And you have to have studies that back that up. You have to be able to say, hey, these are folks that used the Peter Thomas Roth Rose Stem Gel and this is the benefits that they saw and, you know, all of these claims about the stem cells and the extra hydration. There has to be some science instead of just thinking, well maybe itll work and then putting on their box and selling it.

Farron Cousins:Well and, you know this, this should be a fairly easy one for the lawyers here to prove. I mean, they can look and see if there is in fact any science to back any of this up. Obviously theres past customers to see if theres anything here. So hopefully this is going to be a slam dunk for the lawyers thats going to result in a pretty decent settlement for the people whove been using these products regularly. Again, when its based on science, science is either there or its not. So again, hopefully this is fairly easy and, you know, if it is, this could be one of those ones that settles probably long before it actually makes it to a trial.

Scott Hardy:Exactly. Now, unless your face is actually part rosebud, then maybe that really would help have a, a real positive impact. But I dont know anybody, even if they smell nice, that it might help with.

Farron Cousins:Right. For more information on this issue, you can follow the link in the description of this video, head over to Top Class Actions, and while youre there, make sure you sign up for their weekly newsletter. Scott Hardy with Top Class Actions, thank you very much for talking with us.

Scott Hardy:Youre welcome. Thanks for your time, Farron.

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Lawsuit Claims Anti-Aging Skin Cream Is Totally Useless - The Ring of Fire Network - The Ring of Fire Network

There is new hope for patients with a rare autoimmune disorder. In mild cases, scleroderma causes areas of hardened skin. But in severe cases, it can also cause deadly hardening of internal organs like the lungs.

A transplant typically used to treat cancer is having remarkable results for patients who had little hope of surviving.

A year ago, Chuck Beschta couldn't walk more than a few minutes without stopping to rest.

"Just going out and doing normal activities outside raking the lawn, mowing the grass, shoveling the driveway, whatever, snow blowing those became impossible," he said.

After months of testing, he was diagnosed with severe scleroderma, which was hardening his skin. But even worse, it was hardening his lungs, making it hard to breathe.

"He was getting worse despite the best therapy we had to offer," University of Wisconsin rheumatologist Dr. Kevin McKown said.

McKown recommended a stem cell transplant newly approved for scleroderma to reboot Beschta's immune system.

"There's a process by which they try to remove the autoreactive immune cells, the cells that are caught in the immune process, and then they infuse that back in and hope that the body will basically take up and graft that immune system," McKown said.

Beschta saw almost immediate results. His skin was softer and his breathing improved. He hopes his scleroderma has been cured.

"I think we can be optimistic, and so far the people who have been followed out as far as 10 years out don't seem to be getting it back," McKown said.

Without a transplant, less than half the patients who have diffuse scleroderma and severe lung disease live 10 years past diagnosis.

Stem cell transplants are commonly used to treat leukemia and lymphoma, cancers that affect the blood and lymphatic system.

MEDICAL BREAKTHROUGHSRESEARCH SUMMARYTOPIC: NEW THERAPY FOR SCLERODERMAREPORT: MB #4698

BACKGROUND: Scleroderma is an autoimmune rheumatic disease where an overproduction of collagen produced in the body tissues causes the skin and internal organs to harden. The symptoms and effects range by person, but some common symptoms include hardened patches of skin (locations on the body vary,) painful and numb-feeling fingers and toes, and sharp internal pain in the esophagus, intestines, heart, lungs, or kidneys. Women are four times as likely to have scleroderma and the onset is between 30 and 50 years of age. However, anyone from infants to the elderly can have scleroderma. Possible risk factors include having certain gene variations as other family members, ethnic groups, exposure to certain medications or drugs, and already having another autoimmune disease, like rheumatoid arthritis, lupus or Sjogren's syndrome. (Source: https://www.scleroderma.org/site/SPageNavigator/patients_whatis.html;jsessionid=00000000.app30132b?NONCE_TOKEN=9B76519DF6B5819859319F0B63B805C9#.XheCGVVKhaQ , https://www.mayoclinic.org/diseases-conditions/scleroderma/symptoms-causes/syc-20351952 )

DIAGNOSING: A physical exam will be conducted as well as a blood test to check for elevated levels of antibodies the immune system produced. The doctor will also take a sample of skin to be tested in the lab. If there are complaints about internal pain, the doctor may run other tests, including imaging, organ function, and other blood tests. (Source: https://www.mayoclinic.org/diseases-conditions/scleroderma/diagnosis-treatment/drc-20351957 )

NEW TECHNOLOGY: A new stem cell transplant that's commonly known to treat cancer is improving the quality and quantity of life for those with scleroderma. Rheumatologists at University of Wisconsin Health tested the treatment since they have already been conducting bone marrow transplants for decades. Surgeons take out a sample of the patient's bone marrow, isolate the stem cells, and use radiation and chemotherapy to clean out their immune system. The same stem cells are later injected back into the patient's immune system with the hope that new cells will grow and the system is rid of the bad ones. The process is dangerous when the cells are taken out because the patient's immune system is more vulnerable, making infections more likely to occur. However, after four and a half years, 79% of patients that underwent the treatment were alive without serious complications compared to 50% that were treated with the original drugs. (Source: https://madison.com/wsj/news/local/health-med-fit/man-with-severe-autoimmune-disease-gets-stem-cell-transplant-at/article_7e8e17a5-21da-52f8-b728-fe584dab2b77.html)

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New technique developed to treat hardening of internal organs - WNDU-TV

INTRODUCTION

Severe combined immunodeficiency (SCID) is a heterogeneous group of genetic diseases characterized by severe T cell lymphopenia, causing increased susceptibility to viral, bacterial, and fungal infections since early in life (1). Most forms of SCID are due to genetic defects that are intrinsic to hematopoietic cells and can be successfully treated by allogeneic hematopoietic stem cell transplantation (HSCT). However, SCID may also be caused by genetic abnormalities that are intrinsic to thymic epithelium development and function; in such cases, thymus transplantation, but not hematopoietic cell transplantation, is required to cure the disease. Only a few genetic abnormalities, including complete DiGeorge syndrome, and pathogenic variants affecting FOXN1 or CHD7, are known to cause SCID as a result of abnormal thymic development in humans (1).

PAX1 is a member of the paired box (PAX) family of transcription factors and plays a critical role in pattern formation during embryogenesis. It is expressed in the pharyngeal pouches that give rise to the thymus, tonsils, parathyroid glands, thyroid, and middle ear development during human embryogenesis (2). Pax1 deficiency in mice is characterized by anomalies of the vertebral column and variable degrees of thymic hypoplasia and thymocyte number and maturation (35). In humans, a homozygous pathogenic PAX1 p.Gly166Val variant (6) and a homozygous frameshift insertion (c.1173_1174insGCCCG) (7) have been identified in patients with otofaciocervical syndrome type 2 (OTFCS2), a rare disorder characterized by facial dysmorphism, external ear anomalies with preauricular pits and hearing impairment, branchial cysts or fistulas, anomalies of the vertebrae and the shoulder girdle, and mild intellectual disability. Recently, another homozygous pathogenic PAX1 variant (p.Cys368*) has been reported in two affected children from a consanguineous family of North African descent, who presented with OTFCS2 associated with T B+ SCID (8). However, limited information was provided on the immunological phenotype of these patients, and the functional consequences of the PAX1 variant were not investigated. Here, we provide an in-depth clinical, biochemical, and immunological description of multiple patients with OTFCS2 associated with SCID who carried biallelic deleterious PAX1 variants. By performing transfection experiments, molecular modeling, molecular dynamics (MD) simulation, and in vitro differentiation of control- and patient-derived induced pluripotent stem cells (iPSCs) to thymic epithelial progenitor (TEP) cells, we sought to assess the effects of human PAX1 deficiency on thymus development and function.

Patient 1 (P1) is a male infant born to parents whose families were from the same rural region in Germany (Fig. 1A). Bilateral microtia, malar prominence, narrow alae nasi, cupid bow lip, and retrognathia were noticed at birth (fig. S1, A and B). Imaging studies demonstrated severely stenotic external auditory canal on the right side and narrow left auditory canal (fig. S1C), congenital kyphosis at C3-C4 and L3 levels, moderate spinal canal narrowing (fig. S1, D to F), and traction on the cauda equina (fig. S1G). Diffuse erythematous rash (fig. S1H), lymphadenopathy, elevated serum immunoglobulin E (IgE), and eosinophilia were present, consistent with Omenn syndrome. On chest x-ray, the thymus shadow was not visible, and split cervical vertebral bodies, hooked distal clavicles, and a shallow dysplastic glenoid fossa were seen (fig. S1I). This infection history during infancy included Staphylococcus aureus bacteremia, pneumonia, cellulitis, and diarrhea due to Clostridium difficile.

(A) Pedigrees and results of Sanger sequencing in patients with PAX1 variants and in healthy controls. For both family A and family B, results of Sanger sequencing in the heterozygous parents are also shown. (B) Schematic representation of the PAX1 protein and location of the variants identified in affected individuals.

P2 and P3 have been previously described (8) as patients V:1 and V:18, respectively, and are part of a large consanguineous family of Moroccan origin (Fig. 1A). At birth, P2 was noticed to have frontal and parietal bossing, hypertelorism, small nose with hypoplastic nasal root, low-set ears with agenesis of the left pinna and hypoplasia of the right pinna, scapular winging, and bilateral cryptorchidism. Imaging studies showed impaired development of internal auditory canals bilaterally and lack of a thymic shadow. P3 manifested similar facial dysmorphisms as P2, along with left facial nerve palsy, severe dorsal and lumbar scoliosis, and deafness. Imaging studies documented lack of thymic shadow, abnormal appearance of vertebrae, clavicles and shoulder blades, narrowing of both external auditory canals (fig. S1J), abnormalities of the middle ear, and presence of tubular structures with features of a dental element behind the mandibular condyle (fig. S1, K and L). Subject V:3 from the same family died early in life with a history of severe infections, but no formal medical records are available.

P4 and P7 are siblings born to consanguineous parents from Saudi Arabia. P7 was noticed to have severe bilateral microtia, postauricular sinuses, and micrognathia. He suffered from chronic diarrhea, recurrent respiratory infections, exfoliative dermatitis, regional dissemination of Bacille Calmette-Guerin (BCG-itis), and lymphadenopathy and died at 1 year of age.

P4 is a female with a history of chronic diarrhea, recurrent respiratory infections, and poor weight gain since the age of 1 month. Physical examination showed small malformed ears, a skin tag on the right ear, facial asymmetry, small nose with depressed nasal bridge, and small almond-shaped eyes. A skeletal survey showed wedge-shaped vertebral body at T11 and deficient posterior element of the sacrum at S4 and S5.

P5 and P6 were siblings born to consanguineous parents and belonged to the same extended family as P4 and P7. P5 had small, low-set malformed ears, triangular mouth, down-slanting palpebral fissures, a small nose with a depressed nasal bridge, and right facial palsy. She developed recurrent respiratory infections, chronic diarrhea, severe exfoliative dermatitis, and BCG-itis and was diagnosed with Omenn syndrome. She died at 8 months of age with progressive severe pneumonitis.

P6 was screened for immunodeficiency at birth because of the positive family history. She had malformed and low-set small ears, small chin, protruding forehead, and generalized eczema. A skeletal survey showed central depression of the vertebral bodies in the thoracic and lumbar spine. Her immunological workup was consistent with T B+ NK+ (natural killerpositive) SCID. She suffered from recurrent respiratory infections and chronic diarrhea and died at 9 months of age with respiratory syncytial virus (RSV) pneumonia.

The main immunological findings at presentation in P1 to P6 are shown in Table 1. In particular, P1 had significant T cell lymphopenia. His CD4+ lymphocytes were largely (98%) CD45R0+, no CD4+ CD45RA+ CD31+ cells were detected, and T cell proliferation to phytohemagglutinin (PHA) was impaired (fig. S2A). T cell receptor (TCR) excision circles (TRECs) were below the limit of detection, indicating lack of thymopoiesis. TCR V spectratyping revealed T cell oligoclonality (fig. S2B). Elevated serum IgE and eosinophilia were present, consistent with an Omenn syndrome presentation.

AEoC, absolute eosinophil count; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; n.d.: not done; cpm, counts per minute.

Laboratory investigations in P2 at 2 weeks of age revealed profound T cell lymphopenia, markedly reduced proliferative response to mitogens, and increased serum IgE. An inguinal lymph node biopsy showed severe lymphoid depletion, with primary follicles without germinal centers, associated with nearly complete absence of CD3+ T cells, but presence of B and NK cells and sparse plasma cells, and increased number of CD68+ histiocytes and eosinophils (fig. S3). A diagnosis of T B+ NK+ SCID was established.

Severe T cell lymphopenia was observed in P3, P4, and P6, associated with virtually absent in vitro T cell proliferation to PHA in P4 and P6, consistent with a diagnosis of T B+ NK+ SCID (Table 1). Last, P5 was diagnosed as having Omenn syndrome based on generalized erythroderma, lymphocytosis, eosinophilia, hypogammaglobulinemia, increased IgE, and severely reduced in vitro T cell proliferation to PHA.

Because of severe immunological abnormalities, HSCT was attempted in P1 to P4 before the gene defect was known. Details of transplant, chimerism, and immune reconstitution are shown in Table 2. In all cases, a conditioning regimen was used. Two patients (P1 and P4) attained full donor chimerism. P2 failed to engraft, developed interstitial pneumonitis, and died 5.5 months after HSCT. In P3, initial engraftment was followed by secondary graft failure, and a second HSCT was performed, resulting in mixed chimerism. Although three of the patients attained either full or mixed donor chimerism, none of them achieved reconstitution of the T cell compartment. In P1, who exhibits full donor chimerism, all T cells have a CD45R0+ phenotype and therefore likely represent donor-derived T cells contained in the graft that have undergone peripheral expansion. P3 attained mixed chimerism but remained with persistent severe T cell lymphopenia. She developed Pneumocystis jiroveci pneumonia, recurrent gastrointestinal infections, and liver failure and died of septic shock at the age of 4 years and 7 months. P4, who attained full chimerism but failed to reconstitute T cells, developed severe autoimmune hemolytic anemia, requiring multiple courses of rituximab and immunosuppressive therapy. Together, these data indicate that HSCT was unable to correct the profound T cell immunodeficiency of this disease.

ATG, anti-thymocyte globulin; PBSC, peripheral blood stem cells; URD, unrelated donor.

Before HSCT, karyotype analysis revealed no cytogenetic abnormalities in P1, P2, and P3. No evidence for copy number variation (CNV) was found by chromosomal microarray analysis in P1, and search for 22q11 deletion in P2 by in situ fluorescence hybridization was negative. No pathogenic variants in any of the known SCID-causing genes were identified in P4 by a targeted next-generation sequencing primary immunodeficiency gene panel. In an attempt to define the molecular mechanisms of the disease, whole-exome sequencing (WES) was performed in P1, P2, and P4 independently (fig. S4 and table S1). In P1, a total of 153,376 variants were identified. Assuming autosomal recessive inheritance, and upon filtering for homozygous, rare, nonsynonymous changes in coding regions and splice sites, 38 variants were considered. Among these, functional annotation identified the PAX1 NM_006192.3 c.463_465del variant, predicted to cause an in-frame deletion of asparagine at position 155 (p.Asn155del) of the PAX1 protein, as the most likely cause of the disease. In P2, 87,423 variants were detected. Assuming an autosomal recessive inheritance, and upon filtering for homozygous, nonsynonymous, and rare (minor allele frequency < 0.01) variants falling in coding regions or splice sites, 18 such variants were considered. Functional filtering of these revealed the PAX1 c.1104C>A variant, predicted to cause a premature termination at codon 368 (p.Cys368*), as the most likely cause of the disease. In P4, 60,772 variants were detected. Upon filtering for homozygous, nonsynonymous, rare (in-house Saudi variant database <0.005) variants, which were restricted to exonic or splice sites, contained in an autozygome region identified on chromosome 20 by high-density genotyping, and shared with P5 and P6, only two variants were identified, including the PAX1 c.439G>C variant, predicted to cause a p.Val147Leu amino acid change.

Sanger sequencing confirmed homozygosity for the suspected pathogenic PAX1 variants in P1 to P6 (Fig. 1A). The Val147 and the Asn155 amino acid residues are in the DNA-binding paired box domain, and the Cys368 residue is in the transactivation domain of the PAX1 protein (Fig. 1B). All these positions are evolutionarily conserved (fig. S5). The scaled CADD (combined annotation dependent depletion) score (CADD-Phred) for the p.Val147Leu, p.Asn155del, and p.Cys368* variants is 28.1, 21.2, and 38, respectively, significantly higher than the mutation significance cutoff (MSC) score (9), which for the PAX1 gene is 12.06. Together, these data strongly support a pathogenic role of the PAX1 variants identified. Of note, while molecular and cellular studies to confirm the pathogenic role of the PAX1 variants were under way, another group independently attempted WES in P3 and in other family members (but not in P2) and reported the occurrence of the p.Cys368* variant in P3 (8).

To examine the effects of the PAX1 variants at the protein level, we transfected 293T cells with plasmids encoding for either wild-type (WT) or mutant PAX1 complementary DNA (cDNA) and analyzed protein expression by Western blot. In this assay, we also included the PAX1 p.Gly166Val variant, which had been previously reported in a patient with OTFCS2 (6). As shown in Fig. 2A, all mutant proteins were expressed at similar levels as WT PAX1, with the p.Cys368* mutant migrating as a lower molecular weight product, as predicted. To check whether the identified variants altered the subcellular localization of the PAX1 protein, 293T cells were transfected with PAX1 constructs with an N-terminal HA tag, and immunofluorescence was performed with tetramethyl rhodamine isothiocyanate (TRITC)conjugated anti-HA antibody. As shown in Fig. 2B, both WT and mutant PAX proteins were detected in the nucleus, indicating that these variants do not affect subcellular localization.

(A) Western blot showing expression of WT and mutant human PAX1 proteins upon transient transfection in 293T cells. (B) Left: Intracellular protein localization upon transfection of HA-tagged WT and mutant PAX1 constructs into 293T cells, followed by staining with TRITC anti-HA. Right: Counterstaining with DAPI, demonstrating that the mutant PAX1 protein retains nuclear translocation capacity. Scale bar, 10 m. (C) Results of a luciferase reporter assay demonstrating reduced transcriptional activity of mutant PAX1 proteins, corresponding to the PAX1 variants detected in patients. The promoter region of Nkx3-2 was used to drive luciferase expression. Results of six independent experiments (each run in triplicate) are shown (means SEM). P value was calculated with one-way ANOVA and adjusted by Dunnetts multiple comparisons test. **P < 0.01; ***P < 0.0001.

Next, we tested the transcriptional activity of the PAX1 mutant proteins. Little is known on transcriptional targets of human PAX1; however, the Nkx3-2 promoter has been identified as a PAX1 target in mice (10). Therefore, we generated a reporter system in which luciferase expression is driven by the mouse Nkx3-2 promoter. In parallel, we generated both WT (Pax1WT) and mutant (Pax1Val138Leu, Pax1Asn146del, Pax1Cys359*, and Pax1Gly157Val) N-terminal HA-tagged mouse Pax1 constructs, which encode for mouse mutant PAX1 proteins corresponding to the human p.Val147Leu, p.Asn155del, p.Cys368*, and p.Gly166Val variants, respectively. Western blot analysis confirmed that the mutant mouse PAX1 proteins were expressed at similar levels as WT PAX1 (fig. S6). Upon cotransfection of the Nkx3.2-luciferase reporter plasmid and of either WT or mutant PAX1 expression plasmids into 293T cells, analysis of luciferase activity showed that the p.Val138Leu, p.Asn146del, and p.Cys359* PAX1 mutant proteins had significantly reduced reporter expression when compared with WT PAX1 (Fig. 2C and data file S1). A similar defect was also observed for the p.Gly157Val mutant, confirming previous findings (6). These data suggest that the human p.Val147Leu, p.Asn155del, and p.Cys368* variants do not affect protein stability or subcellular localization but alter PAX1 transcriptional activity.

The structure of the human PAX1 protein has not been solved experimentally. However, a crystal structure is available for the paired box domain of the highly homologous PAX6 protein (11). Sequence alignment between the paired box domain of PAX6 and PAX1 proteins reveals a high level of conservation with a similarity of 71%, with a 100% coverage of the region to be modeled as calculated with the BLOSUM80 matrix from PSI-BLAST (E = 1.3691 1020). As reported by Kelm et al. (12), this degree of homology often yields a model for the target (PAX1) with an accuracy of less than 1 root mean square deviation (RMSD) of atomic mobility to the experimentally solved structure of the template (PAX6). Because the p.Val147Leu and p.Asn155del mutants fall within the paired box domain of the protein, we assessed whether the reduced functional activity of the mouse p.Val138Leu and p.Asn146del (and by inference, the human p.Val147Leu and p.Asn155del) variants results from an altered structure and/or abnormal DNA binding. To do this, we first developed a structural model of the paired box domain of WT and mutant PAX1 bound to DNA, based on its homology to the published crystal structure of PAX6 [Protein Data Bank (PDB): 6PAX] (11) by the satisfaction of spatial restraints method using Modeler (13). Structural alignment revealed that the paired box domains of the PAX1 and PAX6 proteins are almost identical with a template modeling (TM) score of 0.99963 and RMSD of 0.08 as measured by the TM align algorithm (14). In addition, the high quality of the model is reflected by the fact that 99% of the residues are in the allowed regions of the (phi) versus (psi) angles of the Ramachandran plot, as shown in fig. S7 (15). Therefore, we used this model to derive a corresponding model for the p.Val147Leu and p.Asn155del variants and for the previously described p.Gly166Val PAX1 variant (6), using in silico site-directed mutagenesis and energy minimization refinement as previously described (16). As shown in Fig. 3A, the paired box domain of all three mutant PAX1 proteins retains a structure composed of two globular domains separated by a linker. These structural models were then used in MD simulations for both their free and DNA-bound forms to define how they differ in both structure and time-dependent dynamic behavior from the canonical WT PAX1 protein.

(A) Molecular modeling of the paired box domain of WT and mutant PAX1 proteins, showing the presence of two globular domain separated by a linker. Note that the asparagine residue at position 155 is adjacent to linker domain, and its deletion results in shortening of the last turn of the third helix in the first globular domain of the paired box domain. (B) Molecular superimposition of WT (in light blue) and mutant PAX1 variants after MD simulation, showing that both the Val147Leu and Asn155del variants predominantly affect the conformation of the C-terminal globular domain, whereas both globular domains are affected by the Gly166Val variant. (C) RMSF values of WT PAX1 and of the Val147Leu, Asn155del, and Gly166Val variants during MD simulations. RMSF values are used here as a measure of the flexibility of different regions of the protein during the MD simulations. The Y axes indicate the magnitude of the fluctuation, whereas the X axes indicate the specific location of each amino acid within the paired box domain.

Because the p.Val147Leu variant is located in the first globular domain, the p.Asn155del is also located in this domain and adjacent to the highly flexible linker, and the p.Gly166Val variant is within the linker, we initially performed 200-ps MD simulations of PAX1 in the absence of DNA to capture potential alterations of the rapid movement of this region of the protein in relationship to the N- and C-terminal helix-loop-helix domains. To gain additional insights into the behavior of the protein, we extended these simulations to 10 ns, in the absence or presence of DNA. When a harmonic restraint is applied to reduce the conformational changes in both globular domains during the 200-ps simulation, the linker is observed to move freely. In this situation, the molecular movement of WT PAX1 paired box domain resembles a barbell-shaped harmonic oscillator, where the globular domains move relative to each other without forming bonds that lock them together in space.

At the end of the 200 ps, in the absence of DNA, the linker of PAX1 shortens and the protein populates a conformational landscape where the globular domains come in close proximity to each other, with the linker located between the N-terminal helix 3 (H3) and the C-terminal helix 1 (H1), respectively (fig. S8). In the most extended conformation of the linker, the interglobular domain distance measured from the Gly158 -C to the Pro175 -C shortens from an original 38.946 to 21.414 (SD = 2.421, P = 0.0001). This shortening contributes to the differences in the RMSD curve, where in the first part of the simulation we observed significant changes due to this shortening, whereas the difference in conformational sampling decreases toward the end of the run. Identical results were obtained in 10-ns simulations. Thus, this H3-Linker-H1 state is likely the one that the PAX1 binding domain adopts when in conformational equilibrium before binding to DNA. In this manner, the linker would be free to contact the minor groove of the DNA and extend in a manner that allows the positioning of both globular domains for full binding. These results led us to set up simulations that would enable gathering information on potential differences in DNA binding among the WT and mutant PAX1 variants.

To investigate whether alterations in the structure or the dynamics of the PAX1 variants have the potential to affect the protein function as a transcription factors, we modeled these proteins in complex with DNA. For this purpose, we again used the bound form of PAX6 as a template. Figure S9 shows the energy-minimized structure of these models before MD simulations. Because the variants identified in the patients either change the sequence of the linker (p.Gly166Val) or the N-terminal globular domain (p.Val147Leu and p.Asn155del), we compared the structures of these variants with WT PAX1 after MD simulation. Because the structure of the DNA interacting with WT or mutant PAX1 proteins was the same in all models shown in fig. S9, we removed it to facilitate the observation of changes that occur in the PAX1 polypeptide chain. When compared with WT PAX1, the p.Val147Leu and the p.Asn155del variants associated with OTFCS2 + SCID differ in particular at the C-terminal second globular domain, as shown by molecular superimposition (Fig. 3B). This result is consistent with the measured root mean square fluctuation (RMSF) values, which shows that the second globular domain is highly flexible in the p.Val147Leu and p.Asn155del mutant proteins (Fig. 3C). By contrast, RMSF values in the first globular domain were lower in all mutant proteins (and especially so in the p.Asn155del and p.Gly166Val mutants) as compared with WT PAX1. Considering these changes, we evaluated potential alterations in the ability of these proteins to recognize and bind to DNA in silico. For this purpose, we analyzed the PAX1-DNA interface. As shown in Fig. 4, as compared with WT PAX1, a lower number of amino acid residues contacting DNA were present within the paired box domain of the p.Val147Leu and p.Asn155del PAX1 mutants. These alterations are more pronounced for the C-terminal region of the domain, which contacts the 3 half of the oligonucleotide and is necessary to maintain appropriate binding to DNA. This altered pattern of interaction with DNA observed in silico may contribute to the altered transcriptional activity of the PAX1 mutant proteins.

Nucleotide residues, in which the paired box domain of either WT or PAX1 mutant proteins establishes interaction, are shown in black. The amino acids contacting nucleotides of target DNA are indicated on the Y axis for each PAX1 protein. The red and green colors indicate loss and gain of DNA binding, respectively.

To gain insights into how pathogenic PAX1 variants may perturb the developmental program of thymic epithelial cells (TECs), we reprogrammed fibroblasts from a healthy control, P1, and P4 to iPSCs and subsequently differentiated these to TEP cells using a previously published protocol (17) with some modifications (see Materials and Methods). Quantitative real-time polymerase chain reaction (qRT-PCR) showed a comparable stemness profile in both control and patient iPSCs (fig. S10), and cytogenetic analysis confirmed their karyotypic integrity. iPSCs were then exposed in vitro to a cocktail of growth factors and molecules that provide essential cues to allow differentiation into definitive endoderm (DE) and eventually into TEP cells (fig. S11A).

To assess changes in the gene expression profile of cells during differentiation, we performed RNA sequencing (RNA-seq) in control cells collected in triplicate at iPS [day 0 (d0)], DE (d5), and TEP (d14) stages of cell differentiation. For each condition, between 15 and 20 million reads were obtained per well. As shown in fig. S11B, during differentiation of control iPSCs to DE and TEPs, we observed progressive changes of gene expression profile, with increased expression of stemness (OCT4, MYC, SOX2, TERT, DNMT3B, and NANOG), endoderm (EOMES, CXCR4, and SOX17), and epithelial (KRT8, CLDN1, EPCAM, LAMA1, and KRT19) genes at iPS, DE, and TEP stages, respectively. In addition, expression of ASXL1, HES1, SHH, GATA3, HOXA3, PSEN1, ZBTB1, HAND2, and MAFB genes, which are all part of the gene set Thymus development, was up-regulated at TEP stage (fig. S11B). Gene set enrichment analysis (GSEA) confirmed differential expression of genes involved in somatic cell maintenance and endoderm development, as well as in other pathways related to differentiation of tissues derived from the third and fourth pharyngeal pouches (fig. S11C).

To assess the reproducibility of the differentiation protocol, we differentiated the same control iPS line twice to TEP cells (named C1 and C2, respectively) in parallel to differentiation of P1 and P4 iPSCs to TEP cells in two distinct differentiation experiments. As shown in Fig. 5A, a similar pattern of changes in the gene expression profile was observed when differentiating control (C1) and P1 iPSCs or control (C2) and P4 iPSCs to TEP cells. In both experiments, control and patient cells showed increased expression of stemness genes at the iPS stage, whereas enhanced expression of epithelial marker genes and of other genes included in the Thymus development gene set was detected at TEP stage. Furthermore, immunohistochemistry analysis confirmed that both control and P1 TEP cells expressed cytokeratin 8 (KRT8), a marker of TECs (fig. S12) (18).

(A) Heatmap of differentially expressed genes between iPS and TEP stage as determined by RNA-seq. Each heatmap shows the top 3000 genes, which were differentially expressed between iPS and TEP cells, with a significance (q < 0.01) by the two-group comparison (t test). Genes whose expression was found to be up-regulated at the TEP stage included epithelial cell markers (EPCAM, KRT8, and KRT19) as well as several genes (PSEN1, HES1, ASXL1, HOXA3, HAND2, EPHB3, and GATA3), which appeared at the leading edge of GSEA of thymus development in (B). (B and C) GSEA on thymus development gene set by preranked genes according to signed log10 adjusted P value. The adjusted P value was acquired by DEseq2 analysis using normalized read count of RNA seq data. FDR, false discovery rate. (D) qRT-PCR analysis of FOXN1 and DLL4 expression at TEP stage of differentiation. Results are from five independent experiments for control and P1, and four independent experiments for control and P4, with triplicates in each case (mean SEM). The P value was calculated with two-tailed paired t test. P < 0.05 was considered to be significant. (E) Thymus development genes with evidence of differential expression between patient and control cells (adjusted P < 0.1 and concordant pattern of expression in both RNA-seq experiments). For this comparison, we considered genes that were part of the Thymus development gene set in MSigDB v7.0, and in the top 30 FOXN1 target genes reported in (19). The values displayed are the signed log10 adjusted P value for differential expression.

GSEA confirmed that upon differentiation of control iPSCs to TEP cells, genes involved in thymus development were more abundantly expressed at the TEP stage both in control and in PAX1 mutant cells (Fig. 5B). Despite similar changes in gene expression profile during differentiation of control- and patient-derived iPSCs to TEP cells, GSEA demonstrated that genes involved in thymus development were more abundantly expressed in control than in patient TEP cells (Fig. 5C). To gain additional mechanistic insights into the severe T cell immunodeficiency of P1 and P4, we performed multiple rounds of differentiation of control and patient iPSCs to TEP cells (five times for control and P1 and four times for control and P4 cells, respectively) and used qRT-PCR to analyze the expression of FOXN1, a master regulator of TEC development (19, 20), and to its target DLL4, a Notch ligand that plays a critical role in T cell commitment (21). FOXN1 expression was significantly reduced in P1 and P4 TEPs as compared with control cells, and a similar trend was observed for DLL4, although the latter significance was reached only when comparing P1 with control TEPs (Fig. 5D and data file S1). Analysis of RNA-seq data revealed several other genes that showed concordantly reduced expression in P1 and P4 TEPs versus control TEPs, reaching statistical significance in at least one of the patients TEP lines (Fig. 5E and table S2). These included STC2, CD83, ZAR1, and ANKMY1, which are known FOXN1 target genes (19); TP63, a regulator of TEC proliferation and aging (22, 23); BMP4, which has been implied in thymus development (24, 25) and in maintenance of TEPs (26, 27); and EYA1 and PAX9, which are involved in patterning of pharyngeal endoderm (28, 29). Together, these data indicate that multiple mechanisms contribute to the thymic defects associated with PAX1 deficiency. Consistent with this, and with the syndromic features manifested by the patients, we observed that several genes included in the Neural crest cell differentiation, Ear development, Cartilage development, Pharyngeal system development, and Skeletal system development gene sets also manifested differential expression in P1 and P4 versus control TEPs (fig. S13).

We have studied six patients from three unrelated families in whom biallelic, loss-of-function PAX1 variants underlie a clinical phenotype characterized by OTFCS2 and severe T cell immunodeficiency. The first example of a biallelic, rare PAX1 variant (p.Gly166Val) in a patient with autosomal recessive OTFCS2 was provided by Pohl et al. (6), who also showed reduced transcriptional activity of the mutant PAX1 protein. However, no data on the patients immunological phenotype were provided. More recently, Patil et al. (7) have described two siblings with a homozygous frameshift PAX1 variant causing OTFCS2; one of them lacked a thymic shadow on chest x-ray. Last, the clinical features of OTFCS2 and SCID have been recently reported by Paganini et al. (8) in two of the patients studied here (P2 and P3), but no immunological or mechanistic characterization was provided.

Several mouse models of PAX1 deficiency, due to distinct variants in the Pax1 gene, have been described, including the undulated (un), undulated extensive (unex), undulated short-tail (unS), and undulated intermediate (un-i) models (30). All of these mutant strains display thymic abnormalities, which are more severe in the unS model (30); however, none of them results in complete athymia. A more profound phenotype, with lack of thymus and parathyroids, associated with craniofacial and skeletal abnormalities, has been observed in Pax9/ mice (31). No cases have been reported of humans with biallelic PAX9 pathogenic variants, and heterozygous PAX9 variants in humans are associated with hypodontia but not with thymic defects (32). Together, these data suggest that the impact of PAX1 and PAX9 on thymus development may be different in humans and mice.

To gain insights into the molecular mechanisms by which PAX1 deficiency may cause syndromic SCID in humans, we have first investigated the expression, subcellular localization, and transactivation activity of PAX1 mutant proteins using transient transfection and luciferase reporter studies. Although transient transfection may result in protein overexpression and therefore cannot be directly compared with protein expression in vivo, the PAX1 p.Val147Leu, p.Asn155del, and p.Cys368* mutant proteins retained the capacity to translocate to the nucleus, and the equivalent murine mutant proteins showed decreased transcription factor activity in vitro. Similar results were obtained for the PAX1 p.Gly166Val (and the mouse equivalent p.Gly157Val) variants, confirming previous observations (6). To further investigate the mechanisms underlying the impaired transcriptional activity of the mutant PAX1 proteins, we have performed structural modeling, using the crystal structure of the PAX6 paired box domain as a template. The results suggest that the structural behavior of the paired box domain (consisting of two globular domains interconnected by a linker) was retained in the p.Val147Leu, p.Asn155del, and p.Gly166Val mutants. MD simulation studies have demonstrated that these variants alter the flexibility of the paired box domain and are predicted to alter binding of PAX1 to its target DNA. Our in silico studies suggest that the mutants differ in their ability to gain or lose binding to distinct nucleotides, with possible impact on the severity of clinical and immunological phenotype. Fine characterization of the molecular mechanisms underlying such heterogeneity will require resolution of the crystal structure of the PAX1 paired box domain and precise identification of its human DNA target sequence(s).

By exposing control- and patient-derived iPSCs to defined differentiation cues, we have successfully differentiated iPSCs to TEPs. Comparison of gene expression profile in control- and patient-derived cells at the TEP stage of in vitro differentiation demonstrated altered expression of genes involved in thymus development in patient cells. In particular, qRT-PCR analysis revealed reduced expression of FOXN1, a master gene of thymus development, and of several FOXN1 target genes, including DLL4. Biallelic FOXN1 pathogenic variants in humans are responsible for a syndromic form of SCID that is the equivalent to what is observed in the nude mouse (33, 34). We have recently reported that FOXN1 haploinsufficiency in humans causes severe T cell lymphopenia at birth (35). The reduced levels of FOXN1 expression observed in patient TEPs (and, by inference, in the patients thymus) may therefore play a direct role in the severe T cell lymphopenia observed in these patients. However, analysis of gene expression profile in patient and control TEPs suggests that other mechanisms, besides reduced FOXN1 expression, may also contribute to impaired thymic development associated with PAX1 deficiency. In particular, reduced TP63 expression may cause impaired TEC proliferation and hence thymic hypoplasia. Moreover, we observed that both P1 and P4 TEPs displayed significantly reduced expression of BMP4 as compared with control TEPs. Conditional deletion of Bmp4 from the pharyngeal endoderm before Foxn1 expression disrupts thymus morphogenesis in mice (24). Furthermore, recent studies have indicated that BMP4 plays a critical role in maintenance of TEC progenitors (27), and reduced BMP4 expression might alter replenishment of the TEC compartment. Future studies based on precise enumeration of TEPs generated in vitro from patient- and control-derived iPSCs may help test this hypothesis. In any case, these data suggest that PAX1 deficiency causes early and more global effects on the development of tissues derived from the third and fourth pharyngeal pouches, including the thymus. Consistent with this hypothesis, patient TEPs were concordant in the abnormal expression of a number of genes involved in skeletal, cartilage, pharyngeal, neural crest, and ear development. Abnormalities in these pathways during differentiation of tissues derived from the third and fourth pharyngeal pouches are likely to contribute to the broad range of malformations observed in the patients reported here.

Last, we have reported that HSCT, which was attempted in four of the six patients, failed to correct the T cell immunodeficiency, despite engraftment in three of them. PAX1 deficiency should be added to the list of severe T cell immunodeficiencies characterized by a primary thymic defect, which also includes complete DiGeorge syndrome, CHARGE syndrome, and FOXN1 deficiency (1). Thymus transplantation represents the treatment of choice to correct the immunodeficiency in these disorders (3638). By contrast, use of unmanipulated HSCT may allow engraftment of donor-derived postthymic T cells that may expand in the recipient, as also observed in P1 in this study, but does not permit de novo generation of a polyclonal repertoire of nave T cells (39). In summary, we have provided mechanistic insights into the pathophysiology of OTFCS2 associated with severe T cell immunodeficiency, an autosomal recessive condition caused by PAX1 variants, and have demonstrated the thymic-intrinsic nature of the immunodeficiency of this condition.

The scope of the study was to identify the molecular basis of a syndromic form of SCID and to perform genomic, molecular, biochemical, structural modeling, and in vitro disease modeling studies to analyze deleterious effects of the PAX1 variants identified. All patients provided written informed consent, according to protocols approved by the local Institutional Review Boards (IRBs). Research studies were performed under National Institutes of Health (NIH) IRB-approved protocol 16-I-N139. For P4, public disclosure of secondary genomic findings was not permitted by the protocol and consent form approved by the local IRB.

WES was performed on P1 and his healthy parents and on P2 and P4 without parental samples. Detailed methods for capture, library preparation, and bioinformatic analysis are described in the Supplementary Materials. Candidate variants were confirmed by Sanger sequencing and described according to Human Genome Variation Society (HGVS) guidelines. For P1 and P2, WES data have been deposited to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) Submission Portal, with the following ID: PRJNA601119.

Flow cytometry studies were performed on either a 10-color Gallios (Beckman Coulter, Brea, CA) or an 8-color Canto II (BD Biosciences, San Jose, CA) cytometer, and results were analyzed using Kaluza software v1.5 (Beckman Coulter, Brea, CA). T cell proliferation studies were performed using Edu-based (Thermo Fisher Scientific, Waltham, MA) flow cytometry method in P1, and tritiated thymidine (3HTdR) incorporation in P2, P4, P5, and P6. TCR V repertoire spectratyping was carried out using a fragment length method on a capillary electrophoresis system (ABI 3730xl DNA Sequencer, Applied Biosystems Inc., Thermo Fisher, Waltham, MA), and data were analyzed using the GeneMarker (v.2.4.0) software (SoftGenetics, State College, PA). All reference values for interpretation were established in the laboratory using healthy pediatric donors recruited via an IRB-approved protocol.

293T cells were plated as 4 105 cells per well in a 12-well plate. After 24 hours, cells were transfected with 1.2 g of pCMV-HA-N vector containing either WT or mutant PAX1 cDNAs, with the Lipofectamine 3000 transfection kit (Thermo Fisher Scientific) following the manufacturers instructions. After 24 hours, cells were collected, lysed, and transferred onto a nitrocellulose membrane. Immunoblotting was performed with rat anti-PAX1/Pax1 monoclonal antibody (mAb) (clone 5A2) (40), followed by staining with horseradish peroxidase (HRP)conjugated goat anti-rat IgG (ab97057; Abcam, Cambridge, MA). After stripping, the membrane was reblotted with rabbit anti-actin mAb (clone 13E5; Cell Signaling Technology, Danvers, MA), followed by Amersham enhanced chemiluminescence anti-rabbit IgG, HRP-linked whole antibody (NA934; GE Healthcare, Helsinki, Finland).

To analyze PAX1 subcellular localization, 293T cells were cultured in polylysine-coated -Slide 8 well (ibidi, Fitchburg, WI) and transfected with 100 ng of pCMV-HA-N vector containing either WT or mutant PAX1 cDNA, with the Lipofectamine 3000 transfection kit (Thermo Fisher Scientific) following the manufacturers instructions. After 24 hours, cells were fixed in 4% paraformaldehyde with phosphate-buffered saline (PBS) for 30 min at room temperature, washed twice in PBS, and then blocked for 1 hour with 10% donkey serum and 0.1% Triton X-100 with PBS at room temperature. Cells were incubated with mouse anti-HA-TRITC mAb (clone H9037; MilliporeSigma, St. Louis) diluted 1:200 in PBS and with 4,6-diamidino-2-phenylindole (DAPI) at room temperature for 1 hour in the dark. Images were obtained with a Leica SP8 (690/730) confocal microscope.

For immunofluorescence analysis of KRT8 expression by TEPs, cells were fixed in 4% paraformaldehyde with PBS for 30 min at room temperature, washed twice in PBS, blocked for 1 hour in 10% donkey serum and 0.1% Triton X-100 with PBS at room temperature, and incubated overnight at 4C with mouse anti-KRT8 antibody (ab2530, C-43) (Abcam, Cambridge, MA) diluted 1:200 in PBS, then for 1 hour at room temperature in the dark with donkey anti-mouse IgG (H+L) Alexa Fluor 488 (ab150105; Abcam) at 1:500 dilution in PBS, and with DAPI (Thermo Fisher Scientific) at 1:1000 dilution in PBS. Images were taken with a Leica SP8 (690/730) confocal microscope.

The promoter region of the mouse Nkx3-2 gene was amplified and cloned into the firefly reporter plasmid pGL4.10 luc2 vector (Promega, Madison, WI), as described (6, 10). To generate expression plasmids containing the mouse Pax1WT, Pax1V138L, Pax1N146del, Pax1G157V, and Pax1C359* coding sequences, the coding sequence of mouse Pax1 (NM_008780.2) was amplified by RT-PCR from isolated adult mouse thymus RNA and cloned into a pCMV-HA-N vector (Addgene, Cambridge, MA) with the In-Fusion HD EcoDry Cloning Kit (Clontech, Mountain View, CA). Pax1 mutant variants were generated by site-directed mutagenesis, and the PCR products were ligated with the Quick Ligation Kit (NEB, Ipswich, MA) and cloned by Turbo competent cells (NEB, Ipswich, MA). The correct sequence of the constructs was confirmed by Sanger sequencing.

The transcriptional activity of WT and mutant PAX1 mouse proteins was assessed in a luciferase reporter assay. 293T cells were cultured in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum with antibiotics and plated in 24-well plates 24 hours before transfection. Transient transfections were performed in triplicate with TransIT-293 Transfection Reagent (Mirus, Madison, WI) according to the manufacturers instructions. Cells were cotransfected with 30 ng of either WT or mutant Pax1 expression plasmids, 15 ng of firefly reporter plasmid Nkx3-2-pGL4.10 luc2, and 3 ng of pRL-TK vector (Promega, Madison, WI) for normalization. After 48 hours, cell extracts were collected and frozen in lysis buffer overnight at 20C. After thawing, firefly and renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI) and Paradigm Detection platform (Beckman Coulter, Indianapolis, IN). To correct for variations in transfection efficiency, firefly luciferase activity was normalized to renilla luciferase activity. The luciferase activity of pCMV-HA-N vector, which had no Pax1 cDNA, was assumed to have 0% activity, whereas the Pax1WT vector was assumed to have 100% activity.

The three-dimensional complex structures of WT and mutant PAX1 models bound to DNA were generated by homology-based methods (16) using the previously solved structure of the highly homologous protein, PAX6 (PDB: 6PAX) (11). Intermolecular interactions of the PAX1 paired box domain of WT/mutant PAX1 to DNA complex were calculated in the Receptor-Ligand function of Discovery Studio Client 4.0 using the default parameters (BIOVIA, San Diego, CA). The MD simulations were performed as described (16).

Primary skin fibroblasts from P1, P4, and a healthy control (BJ fibroblast line, American Type Culture Collection) were reprogrammed to iPSCs by infection with the nonintegrating CytoTune Sendai viral vector kit (Thermo Fisher Scientific) as described (41).

For differentiation, iPSCs were transferred to plates coated with Corning Matrigel human embryonic stem cell (hESC)qualified Matrix. After four to five passages, the cells were plated on Matrigel-coated 24-well plates at a density of 2.5 105 cells/cm2. For differentiation to DE and TEPs, iPSCs were exposed to various factors and differentiation cues, according to the protocol by Parent et al. (17), with some modifications. In particular, between d1 and d5, iPSC differentiation was carried out in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 1% penicillin/streptomycin, 1% l-glutamine, and increasing concentrations of KSR (0% on d1, 0.2% on d2 and d3, and 2% on d4 and d5). In the period d6 to d14, cells were differentiated in DMEM/F12 with 1% penicillin/streptomycin, 1% l-glutamine, and 0.5% (v/v) B-27 supplement (Thermo Fisher Scientific, Waltham, MA). During this period of time, the following factors were added to the culture: activin A, 100 ng/ml (d1 to d5); Wnt3a, 25 ng/ml (d1) or 50 ng/ml (d8 to d14); all-trans retinoic acid (RA), 0.25 M (d6 to d8) or 0.1 M (d9 to d14); BMP4, 50 ng/ml (d6 to d14); LY364947, 5 mM (d6 to d9); FGF8b, 50 ng/ml (d8 to d14); and KAAD-cyclopamine, 0.5 mM (d8 to d14). Supplements and factors were from Thermo Fisher Scientific, Waltham, MA (B27, KSR); R&D Systems, Minneapolis, MN (activin A, Wnt3a, BMP4, and FGF8b); and MilliporeSigma, St. Louis, MO (RA, KAAD-cyclopamine, LY364947).

Microgram quantities of total RNA were isolated using the RNeasy Kit (QIAGEN, Hilden, Germany) from triplicate samples of control-, P1-, and P4-derived iPSCs, as well as from the corresponding iPSC-derived cells at DE and TEP stages. RNA integrity was tested by microfluidic electrophoresis on a TapeStation system (Agilent, Santa Clara, CA). RNA purity and concentration were assessed using the NanoDrop One UV-Vis Spectrophotometer (Thermo Fischer Scientific, Waltham, MA). Directional, mRNA-seq libraries for experiment 1 were produced using TruSeq Stranded mRNA Library Prep Kit for NeoPrep (catalog no. NP-202-1001) from Illumina (San Diego, CA). Directional, mRNA-seq libraries for experiment 2 were produced using New England Biolabs product NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog no. E7490L), New England Biolabs product NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog no. E7760L), and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (catalog no. E7600S) (New England Biolabs, Ipswich MA), with an input of 100 ng of total RNA per sample.

Sequencing was performed on an Illumina NextSeq 500 system, running Illumina NextSeq Control Software System Suite version 2.1.2 and RTA version 2.4.11. The final library pool was sequenced via 1 75base pair (bp) run configuration using the product NextSeq 500/550 High Output v2 sequencing kit, 75 cycles (catalog no. FC-404-2005). Between 15 106 and 20 106 reads were obtained from each sample. RNA-seq FASTQ files were aligned to the reference human genome assembly (GRCh38) with STAR v2.6.0 (42). The transcript annotation (GTF) file was obtained from GENCODE (release 28) (43). The binary alignment files (.bam) were then used to generate a matrix of read counts with the featureCounts program of the package Subread v.1.6.2 (44). Exonic fragments were grouped at the level of genes, based on the GENCODE 28 annotation file. Normalization and differential expression analysis for RNA-seq data were performed with the DESeq2 (45) package in R (46).

Independent pairwise analyses were performed on triplicate samples of cells at each stage of differentiation (iPSC, DE, and TEP). To handle the lower power associated with small numbers of samples, DESeq2 uses an empirical Bayesian procedure to stabilize the log fold change estimates. The Wald test was then applied to the log fold change in each gene, followed by multiple-testing adjustment with the method of Benjamini and Hochberg (47).

For the heatmap of gene expression, t test and hierarchical clustering were computed by Qlucore Omics Explorer 3.3 (Qlucore, Lund, Sweden) for iPSC and TEP stage comparison (Fig. 5A), with cutoff q values of less than 0.01. Analysis of variance (ANOVA) and hierarchical clustering were used for the three-stage (iPSC, DE, and TEP) comparison (fig. S11B). Normalization and differential expression analysis of the RNA-seq data used for GSEA were performed with DESeq2 package in R v.3.5.1. RNA-seq data have been uploaded to the NCBI Gene Expression Omnibus (GEO), under accession no. GSE138784.

GSEA was performed with the GSEA software (48) (http://www.broadinstitute.org/gsea) using a preranked dataset of gene expression differences, 1000 permutations, and the softwares classic enrichment statistic option. Genes were ranked based on the DESeq2 output by taking the signed log10 adjusted P value for differential expression. Gene sets for enrichment analysis correspond to Gene Ontology (GO) Biological Processes and were obtained from the Molecular Signatures Database version 7.0 (GMT file: c5.bp.v7.0.symbols.gmt).

RNA was isolated from control, P1, and P4 cells at iPSC and TEP stages of differentiation, using RNeasy kit (QIAGEN, Hilden, Germany). cDNA was synthesized by a qScript cDNA Synthesis kit (Quantabio, Beverly, MA) according to the manufacturers protocol. qRT-PCR was performed on a 7500 RT-PCR system (Applied Biosystems, Waltham, MA) using PerfeCTa SYBR Green FastMix, Low ROX (Quantabio, Beverly, MA). Gene expression was quantified by normalization to the housekeeping gene TBP for each sample. Primers used for individual genes are reported in the Supplementary Materials.

Statistical analysis was undertaken in GraphPad Prism (v8.0). For luciferase reporter assay, P values were calculated with one-way ANOVA and adjusted by Dunnetts multiple comparisons test. The data are means SEM of six independent experiments (WT, n = 6; Val138Leu, n = 3; Asn146del, n = 5; Cys359*, n = 5; Gly157Val, n = 5; empty, n = 6). For qRT-PCR data, Students t test (paired, two-tailed) was performed. The data are means SEM in Fig. 5D, and means SD in fig. S10. P < 0.05 was considered to be significant. Statistical analysis of RNA-seq data is described above.

Acknowledgments: We thank E. Thorland for interpretive assistance with the CNV analysis and B. Bigio for uploading WES data. WES data have been deposited to the NCBI SRA Submission Portal, with the following ID: PRJNA601119. RNA-seq data have been uploaded to the NCBI GEO, under accession no. GSE138784. Funding: This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), NIH and by the Angelo Nocivelli Foundation. Y.Y. was supported by JSPS Research Fellowship for Japanese Biomedical and Behavioral Research at the NIH and had travel support from The ITO Foundation for the Promotion of Medical Science. R.U. was supported by NIH/NIDDK R01 DK52913, Advancing a Healthier Wisconsin (AHW) Endowment and the Linda T. and Johm A. Mellowes Endowed Innovation and Discovery Fund. L.M.F. is funded by the Division of Intramural Research of the National Institute of Arthritis, Musculoskeletal and Skin Diseases, at the National Institutes of Health. A.A. is supported by King Abdulaziz City for Science and Technology. Author contributions: Y.Y. performed experiments and wrote the manuscript. R.U. performed structural modeling and MD simulation studies. L.M.F. supervised analysis of RNA-seq and GSEA data. F.O.-C., T.G.M., and S. Ganesan assisted with RNA-seq studies. S. Giliani and S.M. performed Sanger sequencing and Western blot analysis and analyzed WES data. K.Z., A.M.A., H.A., F.Z., C.A.V., and B.B. performed and analyzed WES. A.K.D. generated iPSCs. A.J., R.W.M., A.H.F., C.A., B.K.A.-S., and H.A.-M. provided clinical care and description of the patients. F.F. performed lymph node pathology. M.P.B., M.L.H., and C.M. performed and interpreted imaging studies. J.L.C. and R.S.A. contributed to supervision of the project and to writing of the manuscript. L.D.N. was responsible for the entire research project and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Fibroblast and iPSC lines from P1 and P4 are available upon request but are contingent upon approval of material transfer agreement by the NIAID, NIH. WES data have been uploaded to the NCBI SRA Submission Portal, with the following ID: PRJNA601119. The RNA-seq dataset for this study has been uploaded to the NCBI GEO, under accession no. GSE138784. The GEO accession includes links to the NCBI SRA database, from which the raw data will be accessible in FASTQ format, under accession no. SRP225226.

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PAX1 is essential for development and function of the human thymus - Science

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