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Archive for the ‘IPS Cell Therapy’ Category

What’s the Catch: The Fountain of Youth – Paste Magazine

Scientist Juan Carlos Izpisua Belmonte from the Salk Institute in La Jolla, California, claims that the aging process may reversible: Our study shows that aging may not have to proceed in one single direction. With careful modulation, aging might be reversed.

Izpisua Belmonte attests that he implemented a new form of gene therapy on mice that were given a genetic disorder called progeria. After six weeks of treatment, the animals looked youngerand not only that, they had straighter spines, better cardiovascular health, healed more quickly when injured and actually lived longer.

How Its Done The rejuvenating treatment performed on the mice manipulates adult cells, such as skin cells, and turns them back into powerful stem cells (similar to what is seen in embryos). These powerhouses are referred to as induced pluripotent stem (iPS) cells and have the ability to multiply and transform into any cell type in the body; in fact, in trial tests, Izpisua Belmonte says iPS cells are being designed to provide organs and limbs for patients. He claims that his latest study is the first to show that the same technique can be used on other cells to rewind the clock and make them look younger. Izpisua Belmonte explains, The treatment involved intermittently switching on the same four genes that are used to turn skin cells into iPS cells. The mice were genetically engineered in such a way that the four genes could be artificially switched on when the mice were exposed to a chemical in their drinking water.

What This Means: This finding at the Salk Institute suggests that aging may not have to proceed in one directionin fact, Izpisua Belmonte states that it may actually be reversible. Although tests have not been conducted on humans yet, he predicts that applications via creams or injections are a decade away.

This rejuvenating treatment may not lead to immortality, but due to a growing body of evidence, scientists at the Salk Institute theorize that aging is driven by an internal genetic clock that actively causes our body to enter a state of decline. In developing this technology, it is hoped that future treatments designed will slow the ticking of this internal clock and ultimately increase life expectancy.

Whats the Catch? Dr. Sidney Chiu, a 5th year resident at the University of Toronto, thinks this information should be taken with a grain of salt: The findings are promising, but nowhere near ready for the front lines of healthcare. These experiments were done in highly controlled settings on genetically modified mice. If this finding were true, it would be worthy of a Nobel Prize because it would be akin to uncovering the Holy Grail. Chiu elaborates, If you can induce iPS cells, you have the basic building blocks to regenerate anything in the body. But this is far beyond any current medical science we have.

There are also numerous issues to address concerning the study: firstly, the mice are bred in labs for these types of tests, so the variables are controlled from the outset to attain desired results. Chiu adds, In the real world, you cannot turn specific genes on and off using treated water on mice in the wild, let alone humans. There isnt one specific gene for aging; I would be cautious about this scientists claims that isolating merely four could unlock the key to anti-aging. Even if we were just talking about reviving skin tissue, if his findings were true, it would be a breakthrough.

Chiu says that while it is technically possible to alter genetic material when humans are in an embryonic state, that wasnt done here (gene editing research in human embryos is currently allowed in Sweden China, and the United Kingdom. The United States doesnt currently have any legal prohibitions against it).

But its not to say that all of this is in the realm of science fiction; Chiu offers knowledge of research being conducted specifically for telomeres and their relationship to aging. Think of telomeres as the plastic caps that protect your shoelaces from fraying. The laces would be our chromosomes, the recipe for making a living thing. In fact, telomeres have an important role; they protect genetic material from damage that could otherwise lead to diseases or cell death. But because the number of cell divisions in telomeres is finite, once they become shorter (in length) and can no longer reproduce, it causes tissues to degenerate and eventually die. It is theorized that this process may contribute to the human aging process. So scientists are trying to find ways to extend the length of telomeres.

Izpisua Belmonte says that chemical approaches (via creams or injections) might be in human clinical trials to rejuvenate skin, bones and muscle within the next decade. However, from his perspective as a frontline healthcare worker, Chiu believes that we may just have to wait a bit longer than that before such innovations are accessible to everyone.

Main Photo by Thomas Rydberg, CC-BY

Tiffany Leigh is a Toronto-based food, travel, and science writer.

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What’s the Catch: The Fountain of Youth – Paste Magazine

A key ion channel may SLACK off in ALS – ALS Research Forum

Slacking off in ALS? Mutant SOD1 may partially close the SLACK ion channel resulting in increased excitability in some neurons (Zhang et al., 2017).[Image: NIGMS.]

Increased activity in the motor cortex of the brain may occur in most forms of ALS (see September 2015 news). But whether this hyperexcitability contributes to the disease remains an open question.

Now, researchers at Yale University make the case that ALS-linked mutant SOD1 may downregulate a key sodium-gated potassium ion channel, known as SLACK, through an apoptosis signal-regulatingkinase1 (ASK1)-based mechanism (Zhang et al., 2017).

The findings may help explain how motor neuron hyperexcitability occurs in ALS. These changes in excitability may contribute to disease pathogenesis and may underlie fasciculations, one of the earliest clinical manifestations of the disease.

The question is whether this pathway is the primary way that SOD1 mutations cause disease, said Steve Vucic of the University of Sydney, who was not involved in the study. If so, [there] is a tremendous opportunity for developing treatments against these kinase pathways.

The study is published on January 24 in the Journal of Neuroscience.

Excitement builds

Neuronal hyperexcitability emerged in recent years as an early and potentially unifying stepin ALS, due to its detection in a number of sporadic and genetic forms. While the evidence is still not yet conclusive, some studies suggest that this prolonged excitation can lead to toxicity, strengthening the case that these changes in excitability may contribute to the disease (Fritz et al., 2013; Hadzipasic et al., 2014).

How hyperexcitability occurs in ALS remains unclear. But a growing number of studies suggest that mutant SOD1 may be involved, at least in some cases of the disease (Wainger et al., 2014; van Zundert et al., 2008).

Researchers at Yale University, led by Leonard Kaczmarek and Arthur Horwich, wondered whether mutant SOD1 could trigger hyperexcitability in motor neurons by downregulating a key membrane-bound ion channel called SLACK (sequence like a calcium-activated K channel), also known as KCNT1 or KNa1.1.

SLACK is a key regulator of excitability that helps neuronsreturn to the resting state upon firing. Its widely expressed in the CNS and its dysfunction has also been implicated in neurological diseases including Fragile X and epilepsy (Barcia et al., 2012; Heron et al., 2012; Martin et al., 2014).

Hyperexcitability in the bag. Researchers use sea slug bag cell neurons to study underlying hyperexcitability mechanisms. [Image: Kabir et al., 2001 under a CC-BY-NC-SA license.]

To investigate this question, first co-authors Yalan Zhang and Weiming Ni turned to the neuronalmodel system, the sea slug Aplysia. The system gained recognition in the 1960s for its role in providing Eric Kandel Nobel Prize-winning insights into learning and memory formation.

The approachinvolves the manipulation and study of bag cell neurons, very large neuroendocrine cells in the sea slugs abdomen that control egg laying. The really big advantage is that, because of their size, you can inject materials into them and then use a very fine microelectrode to record changes in excitability, all without any disturbance of the cytoplasm, Kaczmarek said.

The researchers compared the activity of potassium channels in bag cell neurons in the presence or absence of wild-type or mutant SOD1, including soluble oligomers of increasing size. They found that SOD1 or mutant SOD1 G85R monomers had no effect. But when they injected SOD1 G85R oligomers, they observed a reduction in outward potassium currents by 20-30%. This drop occured within 10 minutes and increased with larger oligomer size.

Whats more, SOD1 G85R oligomers increased excitability of these neurons. Injection of these soluble 300 kDa protein complexes decreased the neurons resting membrane potential and increased its susceptibility to firing in response to applied stimuli, they found.

Further experiments identified the SLACK channel as the one most likely to have been affected by mutant SOD1, because neurons pretreated with siRNA against SLACK mitigated the effect of these protein complexes in these neurons.

Together, the results suggest that soluble mutant SOD1 oligomericcomplexes may lead to hyperexcitability due to partial closure of SLACK, a key sodium-gated potassium channel that helps neurons return to their resting state upon firing.

ASK1ing for trouble

How could mutant SOD1 downregulateSLACK? The researchers suspected that these effects may be triggered by ASK1, a key kinase that has been previously implicated in the destruction of motor neurons in the disease (Raoul et al., 2002).

ASK1 has been shown to mediate key effects of mutant SOD1 in mouse models of the disease including ER stress and disruption of axonal transport (Lee et al., 2016; Song et al., 2013). In addition, inhibitingthis pathway appears to extend the survival of a SOD1 G93A mouse model of the disease (Fujisawa, et al. 2016).

To investigate this possibility, the researchers blocked ASK1 signaling and determined the impact of SOD1 oligomeric complexes on potassium channel activity. They found that the suppression of outward potassium current could be abolished by pre-treatment with an inhibitor of the apoptosis signaling regulating kinase ASK1. Similar effects were achieved with an inhibitor of one of ASK1s downstream targets, JNK.

The results, Kaczmarek said, suggest that mutant SOD1 oligomericcomplexessuppressSLACK channels in neurons through a ASK1-based mechanism, causing hyperexcitability.

Its an attractive idea, says Massachusetts General Hospitals Brian Wainger, who was not involved in the study. The findings may provide a potentially direct mechanistic connection between mutant SOD1 and motor neuron hyperexcitability in ALS.

Mind your Potassium and KCNQs. Researchers are evaluating Kv 7.2 potassium channel activators including retigabine (orange) in hopes to reduce hyperexcitability in people with the disease. More specific channel modulators are being developed. One such activator, AUT00063, is being evaluated at the phase 2a stage by the London startup Autifony Therapeutics to treat hearing disorders. [Miceli et al., 2011 under CC BY 4.0 license.]

But a change in excitability may not be the only or even the most important consequence of SLACK down regulation, according to Kaczmarek. SLACK may act as an activity sensor, providing a direct link between neuronal firing and protein synthesis.

His teamhas previously shown that SLACK channel activity plays a role in synaptic development, through its ability to regulateactivity-dependent protein synthesis (Brown et al., 2010; Zhang et al., 2012). When you precipitate the channel from mammalian brain, it pulls down several messenger RNAs, he pointed out, and mutations that cause channel overactivity are associated with epilepsy (Barcia et al., 2012; Kim et al., 2015).

In fact, Kaczmarek added, it may not be the hyperexcitability of motor neurons that is toxic in ALS, but rather its proposed (but not yet tested) consequences on protein synthesis. A rapid change in the activity of these channels, as we saw here, is likely going to alter protein synthesis, and that can produce much longer-lasting effects, potentially more consistent with a late-onset disease.

This was an extremely elegant study, and an ingenious way to approach the issue of hyperexcitability, said Steve Vucic, who, in collaboration with University of Sydneys Matthew Kiernan in Australia helped identify these neuronal changes as an early sign of ALS in people with the disease. The goal now will be to see if this same pathway is affected in the mammalian models, or in human ALS iPS cells.

Brian Wainger agrees. The key questions, according to Wainger, are whether these findings hold up in mammalian models, and whether these findings can be generalized to other forms of the disease.

Searching for ALS-linked gene variants in SLACK or related ion channels might also provide insight into its relevance for the human disease, added Vucic.

Approaching the clinic

Hyperexcitability is clearly a clinical feature of many forms of familial and sporadic ALS, explains Wainger. Thats why it is attractive as a convergent mechanism for many forms of ALS. But one of the challenges is to determine to what extent an increase in firing is relevant for disease pathogenesis, rather than, as some argue, being a compensatory mechanism. Directly modulating excitability is one of the clearest ways of answering that question directly, he added.

If motor neuron hyperexcitabilitydoes hold up as a driver of disease, however, it may be a good target for therapy, according to Kaczmarek. I see this as very much a therapeutic possibility.

The reason is because opening up these potassium ion channels may help motor neurons in people with ALS return to their resting state and thereby, reduce hyperexcitability in the disease.

Finding magneto. Researchers are using transcranial magnetic stimulation to evaluate in part whether mexiletine and retigabine reduce hyperexcitability in people with the disease.[Image: NIH].

Kaczmareks team is now hoping to do just that by developing a SLACK activator. The project is ongoing.

In the meantime, clinicians are aiming to reduce hyperexcitability in people with ALS by repurposing existing medicines in hopes to treat the disease. Brian Wainger is leading an effort to determine whether the epilepsy drug retigabine may be helpful in ALS. The drug, identified by Wainger as a potential treatment while in the laboratory of Kevin Eggan, may help normalize the activity of motor neurons by opening up Kv7 potassium channels in people with the disease (see April 2016 news; ; Wainger et al., 2014).

Across the US, the University of Washingtons Michael Weiss is taking a different approach. He is evaluating whether mexiletine, a sodium channel blocker, may reduce hyperexcitability in people with the disease (see March 2016 news). Both strategies are currently at the phase 2 stage.

In a disease that has a selective neuronal vulnerability like ALS, says Wainger, I think it is likely that the electrophysiological properties of the neuron are going to be related to the degenerative nature of the disease. So normalizing those properties may have a good chance of being helpful.

References

Zhang Y, Ni W, Horwich AL, Kaczmarek LK. AnALS-associatedmutantSOD1rapidlysuppressesKCNT1 (Slack) Na+-activated K+ channels in Aplysia neurons. J Neurosci. 2017 Jan 24. pii: 3102-16. [PubMed]

Fritz E, Izaurieta P, Weiss A, Mir FR, Rojas P, Gonzalez D, Rojas F, Brown RH Jr, Madrid R, van Zundert B. MutantSOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability. J Neurophysiol. 2013 Jun;109(11):2803-14. 2013 Mar 13. [PubMed].

Hadzipasic M, Tahvildari B, Nagy M, Bian M, Horwich AL, McCormick DA. Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS. Proc Natl Acad Sci U S A. 2014 Nov 25;111(47):16883-8. [PubMed].

Wainger BJ, Kiskinis E, Mellin C, Wiskow O, Han SS, Sandoe J, Perez NP, Williams LA, Lee S, Boulting G, Berry JD, Brown RH Jr, Cudkowicz ME, Bean BP, Eggan K, Woolf CJ.Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 2014 Apr 10;7(1):1-11.[PubMed]

van Zundert B, Peuscher MH, Hynynen M, Chen A, Neve RL, Brown RH Jr, Constantine-Paton M, Bellingham MC. Neonatal neuronal circuitry shows hyperexcitable disturbance in a mouse model of the adult-onset neurodegenerative disease amyotrophic lateral sclerosis. J Neurosci.2008 Oct 22;28(43):10864-74. [PubMed].

Barcia G, Fleming MR, Deligniere A, Gazula VR, Brown MR, Langouet M, Chen H, Kronengold J, Abhyankar A, Cilio R, Nitschke P, Kaminska A, Boddaert N, Casanova JL, Desguerre I, Munnich A, Dulac O, Kaczmarek LK, Colleaux L, Nabbout R. De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy. Nat Genet. 2012 Nov;44(11):1255-9. [PubMed].

Heron SE, Smith KR, Bahlo M, Nobili L, Kahana E, Licchetta L, Oliver KL, Mazarib A, Afawi Z, Korczyn A, Plazzi G, Petrou S, Berkovic SF, Scheffer IE, Dibbens LM. Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet. 2012 Nov;44(11):1188-90. [PubMed].

Martin HC, Kim GE, Pagnamenta AT, Murakami Y, Carvill GL, Meyer E, Copley RR, Rimmer A, Barcia G, Fleming MR, Kronengold J, Brown MR, Hudspith KA, Broxholme J, Kanapin A, Cazier JB, Kinoshita T, Nabbout R; WGS500 Consortium., Bentley D, McVean G, Heavin S, Zaiwalla Z, McShane T, Mefford HC, Shears D, Stewart H, Kurian MA, Scheffer IE, Blair E, Donnelly P, Kaczmarek LK, Taylor JC. Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis. Hum Mol Genet. 2014 Jun 15;23(12):3200-11. [PubMed].

Raoul C, Estvez AG, Nishimune H, Cleveland DW, deLapeyrire O, Henderson CE, Haase G, Pettmann B. Motoneuron death triggered by a specific pathway downstream of Fas. potentiation by ALS-linked SOD1 mutations. Neuron. 2002 Sep 12;35(6):1067-83. [PubMed].

LeeS, Shang Y, Redmond SA, Urisman A, Tang AA, Li KH, Burlingame AL, Pak RA, Jovii A, Gitler AD, Wang J, Gray NS, Seeley WW, Siddique T, Bigio EH,LeeVM, Trojanowski JQ, Chan JR, Huang EJ. Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis. Neuron. 2016 Jul 6;91(1):41-55. [PubMed].

Song Y, Nagy M, Ni W, Tyagi NK, Fenton WA, Lpez-Girldez F, Overton JD, Horwich AL, Brady ST. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm. Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5428-33. [PubMed].

Fujisawa T, Takahashi M, Tsukamoto Y, Yamaguchi N, Nakoji M, Endo M, Kodaira H, Hayashi Y, Nishitoh H, Naguro I, Homma K, Ichijo H. The ASK1-specific inhibitors K811 and K812 prolong survival in a mouse model of amyotrophic lateral sclerosis. Hum Mol Genet. 2016 Jan 15;25(2):245-53. [PubMed].

Brown MR, Kronengold J, Gazula VR, Chen Y, Strumbos JG, Sigworth FJ, Navaratnam D, Kaczmarek LK. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack. Nat Neurosci. 2010 Jul;13(7):819-21. [PubMed].

Zhang Y, Brown MR, Hyland C, Chen Y, Kronengold J, Fleming MR, Kohn AB, Moroz LL, Kaczmarek LK. Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels. J Neurosci. 2012 Oct 31;32(44):15318-27. [PubMed].

Kim GE, Kronengold J, Barcia G, Quraishi IH, Martin HC, Blair E, Taylor JC, Dulac O, Colleaux L, Nabbout R, Kaczmarek LK. Human slack potassium channel mutations increase positive cooperativity between individual channels. Cell Rep. 2014 Dec 11;9(5):1661-72. [PubMed].

disease-als hyperexcitability mexiletine retigabine SOD1 topic-preclinical topic-researchmodels

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A key ion channel may SLACK off in ALS – ALS Research Forum

Hello, again, Dolly – The Economist

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Hello, again, Dolly – The Economist

Market Players Developing iPS Cell Therapies

While a number of companies have dabbled in this space, the following players are facilitating the development of iPS cell therapies: Cellular Dynamics International (CDI),Cynata Therapeutics, RIKEN, and Astellas (previously Ocata Therapeutics).

While each iPS cell therapy group is considered in detail below, Cellular Dynamics International (CDI) is featured first, because it dominates the iPSC industry. CDI also recently split into two business units, a Life Science Unit and a Therapeutics Unit, demonstrating a commercial strategy for its iPS cell therapy development.

Founded in 2004 and listed on NASDAQ in July 2013, Cellular Dynamics International (CDI) is headquartered in Madison, Wisconsin. The company is known for itsextremely robust patent portfolio containing more than 900 patents.

According to the company, CDI is the worlds largest producer of fully functional human cells derived from induced pluripotent stem (iPS) cells.[1] Their trademarked, iCell Cardiomyocytes, derived from iPSCs, are human cardiac cells used to aid drug discovery, improve the predictability of a drugs worth, and screen for toxicity. In addition, CDI provides: iCell Endothelial Cells for use in vascular-targeted drug discovery and tissue regeneration, iCell Hepatocytes, and iCell Neurons for pre-clinical drug discovery, toxicity testing, disease prediction, and cellular research.[2]

Induced pluripotent stem cells were first produced in 2006 from mouse cells and in 2007 from human cells, by Shinya Yamanaka at Kyoto University,[3] who also won the Nobel Prize in Medicine or Physiology for his work on iPSCs.[4] Yamanaka has ties toCellular Dynamics International as a member of the scientific advisory board of iPS Academia Japan. IPS Academia Japan was originally established to manage the patents and technology of Yamanakas work, and is now the distributor of several of Cellular Dynamics products, including iCell Neurons, iCell Cardiomyocytes, and iCell Endothelial Cells.[5]

Importantly, in 2010 Cellular Dynamics became the first foreign company to be granted rights to use Yamanakas iPSC patent portfolio.Not only has CDI licensed rights to Yamanakas patents, but it also has a license to use Otsu, Japan-based Takara Bios RetroNectin product, which it uses as a tool to produce its iCell and MyCell products.[6]

Furthermore, in February 2015, Cellular Dynamics International announcedit would be manufacturing cGMP HLA Superdonor stem cell lines that will support cellular therapy applications through genetic matching.[8] Currently, CDI has two HLA superdonor cell lines that provide a partial HLA match to approximately 19% of the population within the U.S., and it aims to expand its master stem cell bank by collecting more donor cell lines that will cover 95% of the U.S. population.[9]The HLA superdonor cell lines were manufactured using blood samples, and used to produce pluripotent iPSC lines, giving the cells the capacity to differentiate into nearly any cell within the human body.

On March 30, 2015, Fujifilm Holdings Corporation announced that it was acquiring CDI for $307 million, allowingCDI tocontinue to run its operations in Madison, Wisconsin, and Novato, California as a consolidated subsidiary of Fujifilm.[14] A key benefit of the merger is that CDIs technology platform enables the production of high-quality fully functioning iPSCs (and other human cells) on an industrial scale, while Fujifilm has developed highly-biocompatible recombinant peptidesthat can be shaped into a variety of forms for use as a cellular scaffoldin regenerative medicinewhen used in conjunction with CDIs products.[15]

Additionally, Fujifilm has been strengthening its presence in the regenerative medicine field over the past several years, including a recent A$4M equity stake in Cynata Therapeutics and anacquisition ofJapan Tissue Engineering Co. Ltd.in December 2014. Most commonly called J-TEC, Japan Tissue Engineering Co. Ltd. successfully launched the first two regenerative medicine products in the country of Japan.According toKaz Hirao, CEO of CDI, It is very important for CDI to get into the area of therapeutic products, and we can accelerate this by aligning it with strategic and technical resources present within J-TEC.

Kaz Hirao also states,For our Therapeutic businesses, we will aim to file investigational new drugs (INDs) with the U.S. FDA for the off-the-shelf iPSC-derived allogeneic therapeutic products. Currently, we are focusing on retinal diseases, heart disorders, Parkinsons disease, and cancers. For those four indicated areas, we would like to file several INDs within the next five years.

Finally, in September 2015, CDI againstrengthened its iPS cell therapycapacity by setting up a new venture, Opsis Therapeutics. Opsis is focused on discovering and developing novel medicines to treat retinal diseases and is apartnership with Dr. David Gamm, the pioneer of iPS cell-derived retinal differentiation and transplantation.

In summary, several key events indicate CDIs commitment to developing iPS cell therapeutics, including:

Australian stem cell company Cynata Therapeutics (ASX:CYP) is taking a unique approachby creating allogeneic iPSC derived mesenchyal stem cell (MSCs)on a commercial scale.Cynatas Cymerus technology utilizes iPSCs provided by Cellular Dynamics International, a Fujifilm company, as the starting material for generating mesenchymoangioblasts (MCAs), and subsequently, for manufacturing clinical-gradeMSCs.According to Cynatas Executive Chairman Stewart Washer who was interviewed by The Life Sciences Report, The Cymerus technology gets around the loss of potency with the unlimited iPS cellor induced pluripotent stem cellwhich is basically immortal.

OnJanuary 19, 2017, Fujifilm took anA$3.97 million (10%) strategic equity stakein Cynata, positioning the parties to collaborate on the further development and commercialisation of Cynatas lead Cymerus therapeutic MSC product CYP-001 for graft-versus-host disease (GvHD). (CYP-001 is the product designation unique to the GVHD indication). The Fujifilm partnership also includes potential future upfront and milestone payments in excess of A$60 million and double-digit royalties on CYP-001 product net sales for Cynata Therapeutics, as well as strategic relationship for potential future manufacture of CYP-001 and certain rights to other Cynata technology.

One of the key inventors of Cynatas technology is Igor Slukvin, MD, Ph.D., Scientific Founder of Cellular Dynamics International (CDI) and Cynata Therapeutics. Dr. Slukvin has released more than 70 publications about stem cell topics, including the landmark article in Cell describing the now patented Cymerus technique. Dr. Slukvins co-inventor is Dr. James Thomson, the first person to isolate an embryonic stem cell (ESC) and one of the first people to create a human induced pluripotent stem cell (hiPSC). Dr. James Thompson was theFounder of CDI in 2004.

There are three strategic connections between Cellular Dynamics International (CDI) and Cynata Therapeutics, which include:

Recently, Cynata received advice from the UK Medicines and Healthcare products Regulatory Agency (MHRA) that its Phase I clinical trial application has been approved, titledAn Open-Label Phase 1 Study to Investigate the Safety and Efficacy of CYP-001 for the Treatment of Adults With Steroid-Resistant Acute Graft Versus Host Disease. It will be the worlds first clinical trial involving a therapeutic product derived from allogeneic (unrelated to the patient) induced pluripotent stem cells (iPSCs).

Participants for Cynatas upcoming Phase I clinical trial will be adults who have undergone an allogeneic haematopoietic stem cell transplant (HSCT) to treat a haematological disorder and subsequently been diagnosed with steroid-resistant Grade II-IV GvHD.The primary objective of the trial is to assess safety and tolerability, while the secondary objective is to evaluate the efficacy of two infusions of CYP-001 in adults with steroid-resistant GvHD.

Using Professor Yamankas Nobel Prize winning achievement of ethically uncontentious iPSCs and CDIs high quality iPSCs as source material, Cynata has achieved two world firsts:

Cynata has also released promising pre-clinical data in Asthma, Myocardial Infarction (Heart Attack), andCritical Limb Ischemia.

There are four key advantages of Cynatas proprietary Cymerus MSC manufacturing platform.Because the proprietary Cymerus technology allows nearly unlimited production of MSCs from a single iPSC donor, there is batch-to-batch uniformity. Utilizing a consistent starting material allows for a standardized cell manufacturing process and a consistent cell therapy product. Unlike other companies involved with MSC manufacturing, Cynata does not require a constant stream of new donors in order to source fresh stem cells for its cell manufacturing process, nor does it require the massive expansion of MSCs necessitated by reliance on freshly isolated donations.

Finally, Cynata has achieved a cost-savings advantage through its uniqueapproach to MSCmanufacturing. Its proprietary Cymerus technology addresses a critical shortcoming in existing methods of production of MSCs for therapeutic use, which is the ability to achieve economic manufacture at commercial scale.

On June 22, 2016, RIKEN announced that it is resuming its retinal induced pluripotent stem cell (iPSC) study in partnership with Kyoto University.

2013 was the first time in which clinical research involving transplant of iPSCs into humans was initiated, led by Masayo Takahashi of the RIKEN Center for Developmental Biology (CDB)in Kobe, Japan. Dr. Takahashi and her team wereinvestigating the safety of iPSC-derived cell sheets in patients with wet-type age-related macular degeneration. Althoughthe trial was initiated in 2013 and production of iPSCs from patients began at that time, it was not until August of 2014 that the first patient, a Japanese woman, was implanted with retinal tissue generated using iPSCs derived from her own skin cells.

A team of three eye specialists, led by Yasuo Kurimoto of the Kobe City Medical Center General Hospital, implanted a 1.3 by 3.0mm sheet of iPSC-derived retinal pigment epithelium cells into the patients retina.[196]Unfortunately, the study was suspended in 2015 due to safety concerns. As the lab prepared to treat the second trial participant, Yamanakas team identified two small genetic changes in the patients iPSCs and the retinal pigment epithelium (RPE) cells derived from them. Therefore, it is major news that theRIKEN Institute will now be resuming the worlds first clinical study involving the use of iPSC-derived cells in humans.

According to the Japan Times, this attempt at the clinical studywill involve allogeneic rather than autologous iPSC-derived cells for purposes of cost and time efficiency.Specifically,the researchers will be developing retinal tissues from iPS cells supplied by Kyoto Universitys Center for iPS Cell Research and Application, an institution headed by Nobel prize winner Shinya Yamanaka. To learn about this announcement, view this article fromAsahi Shimbun, aTokyo- based newspaper.

In November 2015 Astellas Pharma announced it was acquiring Ocata Therapeutics for $379M. Ocata Therapeutics is a biotechnology company that specializes in the development of cellular therapies, using both adult and human embryonic stem cells to develop patient-specific therapies. The companys main laboratory and GMP facility is in Marlborough, Massachusetts, and its corporate offices are in Santa Monica, California.

When a number of private companies began to explore the possibility of using artificially re-manufactured iPSCs for therapeutic purposes, one such company that was ready to capitalize on the breakthrough technology was Ocata Therapeutics, at the time called Advanced Cell Technology. In 2010, the company announced that it had discovered several problematic issues while conducting experiments for the purpose of applying for U.S. Food and Drug Administration approval to use iPSCs in therapeutic applications. Concerns such as premature cell death, mutation into cancer cells, and low proliferation rates were some of the problems that surfaced. [17]

As a result, the company shifted its induced pluripotent stem cell approach to producingiPS cell-derived human platelets, as one of the benefits of a platelet-based product is that platelets do not contain nuclei, and therefore, cannot divide or carry genetic information. While the companys Induced Pluripotent Stem Cell-Derived Human Platelet Program received a great deal of media coverage in late 2012, including being awarded the December 2012 honor of being named one of the 10 Ideas that Will Shape the Yearby New Scientist Magazine,[178] unfortunately the company did not succeed in moving the concept through to clinical testing in 2013.

Nonetheless, Astellas is clearly continuing to develop Ocatas pluripotent stem cell technologies involving embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells). In a November 2015 presentation by Astellas President and CEO, Yoshihiko Hatanaka, he indicated that the company will aim to develop an Ophthalmic Disease Cell Therapy Franchise based around its embryonic stem cell (ESC) and induced pluripotent stem cell (iPS cell) technology. [19]

Footnotes [1] CellularDynamics.com (2014). About CDI. Available at: http://www.cellulardynamics.com/about/index.html. Web. 1 Apr. 2015. [2] Ibid. [3] Takahashi K, Yamanaka S (August 2006).Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell126(4): 66376. [4] 2012 Nobel Prize in Physiology or Medicine Press Release. Nobelprize.org. Nobel Media AB 2013. Web. 7 Feb 2014. Available at: http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/press.html. Web. 1 Apr. 2015. [5] Striklin, D (Jan 13, 2014). Three Companies Banking on Regenerative Medicine. Wall Street Cheat Sheet. Retrieved Feb 1, 2014 from, http://wallstcheatsheet.com/stocks/3-companies-banking-on-regenerative-medicine.html/?a=viewall. [6] Striklin, D (2014). Three Companies Banking on Regenerative Medicine. Wall Street Cheat Sheet [Online]. Available at: http://wallstcheatsheet.com/stocks/3-companies-banking-on-regenerative-medicine.html/?a=viewall. Web. 1 Apr. 2015. [7] Cellular Dynamics International (July 30, 2013). Cellular Dynamics International Announces Closing of Initial Public Offering [Press Release]. Retrieved from http://www.cellulardynamics.com/news/pr/2013_07_30.html. [8] Investors.cellulardynamics.com,. Cellular Dynamics Manufactures Cgmp HLA Superdonor Stem Cell Lines To Enable Cell Therapy With Genetic Matching (NASDAQ:ICEL). N.p., 2015. Web. 7 Mar. 2015. [9] Ibid. [10] Cellulardynamics.com,. Cellular Dynamics | Mycell Products. N.p., 2015. Web. 7 Mar. 2015. [11]Sirenko, O. et al. Multiparameter In Vitro Assessment Of Compound Effects On Cardiomyocyte Physiology Using Ipsc Cells.Journal of Biomolecular Screening18.1 (2012): 39-53. Web. 7 Mar. 2015. [12] Sciencedirect.com,. Prevention Of -Amyloid Induced Toxicity In Human Ips Cell-Derived Neurons By Inhibition Of Cyclin-Dependent Kinases And Associated Cell Cycle Events. N.p., 2015. Web. 7 Mar. 2015. [13] Sciencedirect.com,. HER2-Targeted Liposomal Doxorubicin Displays Enhanced Anti-Tumorigenic Effects Without Associated Cardiotoxicity. N.p., 2015. Web. 7 Mar. 2015. [14] Cellular Dynamics International, Inc. Fujifilm Holdings To Acquire Cellular Dynamics International, Inc.. GlobeNewswire News Room. N.p., 2015. Web. 7 Apr. 2015. [15] Ibid. [16]Cyranoski, David. Japanese Woman Is First Recipient Of Next-Generation Stem Cells. Nature (2014): n. pag. Web. 6 Mar. 2015. [17] Advanced Cell Technologies (Feb 11, 2011). Advanced Cell and Colleagues Report Therapeutic Cells Derived From iPS Cells Display Early Aging [Press Release]. Available at: http://www.advancedcell.com/news-and-media/press-releases/advanced-cell-and-colleagues-report-therapeutic-cells-derived-from-ips-cells-display-early-aging/. [18] Advanced Cell Technology (Dec 20, 2012). New Scientist Magazine Selects ACTs Induced Pluripotent Stem (iPS) Cell-Derived Human Platelet Program As One of 10 Ideas That Will Shape The Year [Press Release]. Available at: http://articles.latimes.com/2009/mar/06/science/sci-stemcell6. Web. 9 Apr. 2015. [19] Astellas Pharma (2015). Acquisition of Ocata Therapeutics New Step Forward in Ophthalmology with Cell Therapy Approach. Available at: https://www.astellas.com/en/corporate/news/pdf/151110_2_Eg.pdf. Web. 29 Jan. 2017.

Excerpt from:
Market Players Developing iPS Cell Therapies

Regulators OK Clinical Trials Using Donor Stem Cells – The Scientist


The Scientist
Regulators OK Clinical Trials Using Donor Stem Cells
The Scientist
WIKIPEDIA, TMHLEEResearchers in Japan who have been developing a cell therapy for macular degeneration received support from health authorities this week (February 1) to begin a clinical trial using donor-derived induced pluripotent stem (IPS) cells …

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Regulators OK Clinical Trials Using Donor Stem Cells – The Scientist

What’s the benefit in making human-animal hybrids? – The Conversation AU

The red shows rat cells in the developing heart of a mouse embryo.

A team of scientists from the Salk Institute in the United States created a stir last week with the announcement that they had created hybrid human-pig foetuses.

The story was widely reported, although some outlets took a more hyperbolic or alarmed tone than others.

One might wonder why scientists are even creating human-animal hybrids often referred to as chimeras after the Greek mythological creature with features of lion, goat and snake.

The intention is not to create new and bizarre creatures. Chimeras are incredibly useful for understanding how animals grow and develop. They might one day be used to grow life-saving organs that can be transplanted into humans.

The chimeric pig foetuses produced by Juan Izpisua Belmonte, Jun Wu and their team at the Salk Institute were not allowed to develop to term, and contained human cells in multiple tissues.

The actual proportion of human cells in the chimeras was quite low and their presence appeared to interfere with development. Even so, the study represents a first step in a new avenue of stem cell research which has great promise. But it also raises serious ethical concerns.

A chimera is an organism containing cells from two or more individuals and they do occur in nature, albeit rarely.

Marmoset monkeys often display chimerism in their blood and other tissues as a result of transfer of cells between twins while still in the womb. Following a successful bone marrow transplantation to treat leukaemia, patients have cells in their bone marrow from the donor as well as themselves.

Chimeras can be generated artificially in the laboratory through combining the cells from early embryos of the same or different species. The creation of chimeric mice has been essential for research in developmental biology, genetics, physiology and pathology.

This has been made possible by advances in gene targeting in mouse embryonic stem cells, allowing scientists to alter the cells to express or silence certain genes. Along with the ability to use those cells in the development of chimeras, this has enabled researchers to produce animals that can be used to study how genes influence health and disease.

The pioneers of this technology are Oliver Smithies, Mario Cappechi and Martin Evans, who received a Nobel Prize in Physiology or Medicine in 2007 for their work.

More recently, researchers have become interested in investigating the ability of human pluripotent stem cells master cells obtained from human embryos or created in the laboratory from body cells, to contribute to the tissues of chimeric animals.

Human pluripotent stem cells can be grown indefinitely in the laboratory, and like their mouse counterparts, they can form all the tissues of the body.

Many researchers have now shown they can make functional human tissues of medical significance from human pluripotent cells, such as nerve, heart, liver and kidney cells.

Indeed, cellular therapeutics derived from human pluripotent stem cells are already in clinical trials for spinal cord injury, diabetes and macular degeneration.

However, since 2007 it has been clear that there is not one type of pluripotent stem cell. Rather, a range of different types of pluripotent stem cells have been generated in mice and humans using different techniques.

These cells appear to correspond to cells at different stages of embryonic development, and therefore are likely to have different properties, raising the question about which source of cells is best.

Creating a chimeras has long been the gold standard used by researchers to determine the potential of pluripotent stem cells. While used extensively in animal stem cell research, chimeric studies using human pluripotent stem cells have proved challenging as few human cells survive in human-animal chimeras.

Although the number of human cells in the chimera was low, the findings by the Salk Institute researchers provide a new avenue to address two important goals. The first is the possibility of creating humanised animals for use in biomedical research.

While it is already possible to produce mice with human blood, providing an invaluable insight into how our blood and immune system functions, these animals rely on the use of human fetal tissue and are difficult to make.

The use of pluripotent stem cells in human-animal chimeras might facilitate the efficient production of mice with human blood cells, or other tissues such as liver or heart, on a larger scale. This could greatly enhance our ability to study the development of diseases and to develop new drugs to treat them.

The second potential application of human-animal chimeras comes from some enticing studies performed in Japan in 2010. These studies were able to generate interspecies chimeras following the introduction of rat pluripotent stem cells into a mouse embryo that lacked a key gene for pancreas development.

As a result, the live born mice had a fully functional pancreas comprised entirely of rat cells. If a similar outcome could be achieved with human stem cells in a pig chimera, this would represent a new source of human organs for transplantation.

While scientifically achieving such goals remains a long way off, it is almost certain that progress in pluripotent stem cell biology will enable successful experimentation along these lines. But how much of this work is ethically acceptable, and where do the boundaries lie?

Many people condone the use of pigs for food or as a source of replacement heart valves. They might also be content to use pig embryos and foetuses as incubators to manufacture human pancreas or hearts for those waiting on the transplant list. But the use of human-monkey chimeras may be more contested.

Studies have shown that early cells of the central nervous system made from human embryonic stem cells can engraft and colonise the brain of a newborn mouse. This provides a proof of concept for possible cellular therapies.

But what if human cells were injected into monkey embryos? What would be the ethical and cognitive status of a newborn rhesus monkey whose brain consists of predominantly human nerves?

It may be possible to genetically engineer the cells so that human cells can effectively grow into replacement parts. But what safeguards do we need to ensure that the human cells dont also contribute to other organs of the host, such as the reproductive organs?

While the announcement of a human-pig chimera may have taken many by surprise, regulators and medical researchers well recognise that chimeric research may raise issues in addition to the those already posed by animal research.

However, rather than call for a blanket ban or restricting funding for this area of medical research, it requires careful case-by-case consideration by independent oversight committees fully aware of animal welfare considerations and recognising existing standards.

For example, The 2016 Guidelines for Clinical Research and Translation from the International Society for Stem Cell Research call for research where human gametes could be generated from human-animal chimeras to be prohibited, but supports research using human-animal chimeras conducted under appropriate review and oversight.

Chimeric research will and needs to continue. But equally scientists involved in this field need to continue to discuss and consider the implications of their research with the broader community. Chimeras can all too readily be dismissed as mythological monsters engendering fear.

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What’s the benefit in making human-animal hybrids? – The Conversation AU

Companies Developing Induced Pluripotent Stem Cell (iPS …

While a number of companies have dabbled in this space, the following players are facilitating the development of iPS cell therapies: Cellular Dynamics International (CDI), RIKEN, Cynata Therapeutics, and Astellas (previously Ocata Therapeutics).

While each iPS cell therapy group is considered in detail below, Cellular Dynamics International (CDI) is featured first, because it dominates the iPSC industry. CDI also recently split into two business units, a Life Science Unit and a Therapeutics Unit, demonstrating a commercial strategy for its iPS cell therapy development.

Cellular Dynamics International (CDI) is headquartered in Madison, Wisconsin, although it provides technical support and sales information from both the United States and Japan. CDI was founded in 2004 and listed on NASDAQ in July 2013. The company had global revenues of $16.7 million in 2014 and currently has 150+ employees. It also has an extremely robust patent portfolio containing more than 800 patents, of which 130 pertain to iPSCs.

According to the company, CDI is the worlds largest producer of fully functional human cells derived from induced pluripotent stem (iPS) cells.[1] Their trademarked, iCell Cardiomyocytes, derived from iPSCs, are human cardiac cells used to aid drug discovery, improve the predictability of a drugs worth, and screen for toxicity. In addition, CDI provides: iCell Endothelial Cells for use in vascular-targeted drug discovery and tissue regeneration, iCell Hepatocytes, and iCell Neurons for pre-clinical drug discovery, toxicity testing, disease prediction, and cellular research.[2] As such, CDIs main role with regard to iPCS therapy development is the production of industrial-scale, clinical-grade iPSCs.

As mentioned previously, induced pluripotent stem cells were first produced in 2006 from mouse cells and in 2007 from human cells, by Shinya Yamanaka at Kyoto University,[3] who also won the Nobel Prize in Medicine or Physiology for his work on iPSCs.[4] Yamanaka has ties toCellular Dynamics International as a member of the scientific advisory board of iPS Academia Japan.

IPS Academia Japan was originally established to manage the patents and technology of Yamanakas work, and is now the distributor of several of Cellular Dynamics products, including iCell Neurons, iCell Cardiomyocytes, and iCell Endothelial Cells.[5] Importantly, in 2010 Cellular Dynamics became the first foreign company to be granted rights to use Yamanakas iPSC patent portfolio.Not only has CDI licensed rights to Yamanakas patents, but it also has a license to use Otsu, Japan-based Takara Bios RetroNectin product, which it uses as a tool to produce its iCell and MyCell products.[6] Through its licenses and intellectual property, CDI currently uses induced pluripotent stem cells to produce human heart cells (cardiomyocytes), brain cells (neurons), blood vessel cells (endothelial cells), and liver cells (hepatocytes), manufacturing them in high quantity, quality, and purity.

These human cells produced by the company are used for both in vitro and in vivo applications that range from basic and applied research to drug discovery research that includes target identification and validation, toxicity testing, safety and efficacy testing, and more. As such, CDI has emerged as a global leader with the ability to generate iPSCs that have the potential to be used for a wide range of research and possibly therapeutic purposes.

In a landmark event with the iPSC market, the company had an initial public offering (IPO) in July of 2013, in which it sold 38,460,000 shares of common stock to the public at $12.00 per share, to raise proceeds of approximately $43 million.[7] This event secured the companys position as the global leader in producing high-quality human iPSCs and differentiated cells in industrial quantities.

In addition, in March of 2013, Celullar Dynamics International and the Coriell Institute for Medical Research announced receiving multi-million dollars grants from the California Institute for Regenerative Medicine (CIRM) for the creation of iPSC lines from 3,000 healthy and diseased donors, a result that will create the worlds largest human iPSC bank.

Not surprisingly, Cellular Dynamics International has continued its innovation, announcing in February of 2015 that it would be manufacturing cGMP HLA Superdonor stem cell lines that will support cellular therapy applications through genetic matching.[8] Currently, CDI has two HLA superdonor cell lines that provide a partial HLA match to approximately 19% of the population within the U.S., and it aims to expand its master stem cell bank by collecting more donor cell lines that will cover 95% of the U.S. population.[9]

The HLA superdonor cell lines were manufactured using blood samples, and used to produce pluripotent iPSC lines, giving the cells the capacity to differentiate into nearly any cell within the human body.

CDI also leads the iPSC market in terms of supporting drug development and discovery. For example, CDIs MyCell products are created using custom iPSC reprogramming and differentiation methods, thereby providing biologically relevant human cells from patients with unique disease-associated genotypes and phenotypes.[10] The companys iCell and MyCell cells can also be adapted to screening platforms and are matched to function with common readout technologies.[11] CDIs products are also used for high-throughput screening,[12] and have been used as supporting data for Investigational New Drug (IND) applications submitted to the Federal Drug Administration (FDA).[13]

On March 30, 2015, Fujifilm Holdings Corporation announced that it was acquiring CDI, in which Fujifilm will acquire CDI through all-cash offer followed by a second step merger. Specifically, Fujifilm will acquire all issued and outstanding shares of CDIs common stock for $16.5 per share or approximately $ 307 million, after which CDI will continue to run its operations in Madison, Wisconsin, and Novato, California as a consolidated subsidiary of Fujifilm.[14]

CDIs technology platform enables the production of high-quality fully functioning iPSCs (and other human cells) on an industrial scale, while Fujifilm has developed highly-biocompatible recombinant peptidesthat can be shaped into a variety of forms for use as a cellular scaffoldin regenerative medicinewhen used in conjunction with CDIs products.[15] Fujifilm has been strengthening its presence in the regenerative medicine field over several years, including by acquiring a majority of shares of Japan Tissue Engineering Co. in December 2014, so while the acquisition was unexpected, it as not fully suprising.

In summary, the acquisition of CDI will allow Fujifilm to gaindominance in the areaof iPS cell-based drug discovery services and will position it to strategically combine CDIs iPS cell technologywithFujifilms expertise in material science and engineering systems, creating a powerhouse within the iPSC market. It is yet to be seen whether Fujifilm will try to commercialize CDIs iPS cell production technologies by making the cells available for clinical use or whether they will choose to focus their attention on iPS cell-based drug discovery services.

In November 2015 Astellas Pharma announced it was acquiring Ocata Therapeutics for $379M. Ocata Therapeutics is a biotechnology company that specializes in the development of cellular therapies, using both adult and human embryonic stem cells to develop patient-specific therapies. The companys main laboratory and GMP facility is in Marlborough, Massachusetts, and its corporate offices are in Santa Monica, California.

When a number of private companies began to explore the possibility of using artificially re-manufactured iPSCs for therapeutic purposes, one such company that was ready to capitalize on the breakthrough technology was Ocata Therapeutics (at the time called Advanced Cell Technology or ACT). In 2010, the company announced that it had discovered several problematic issues while conducting experiments for the purpose of applying for U.S. Food and Drug Administration approval to use iPSCs in therapeutic applications. Concerns such as premature cell death, mutation into cancer cells, and low proliferation rates were some of the problems that surfaced. [16]

As a result, the company has since shifted its induced pluripotent stem cell approach to producingiPS cell-derived human platelets, as one of the benefits of a platelet-based product is that platelets do not contain nuclei, and therefore, cannot divide or carry genetic information. Although nothing is completely safe, iPS cell-derived platelets are likely to be much safer than other iPSC therapies, in which uncontrolled proliferation is a major concern.

While the companys Induced Pluripotent Stem Cell-Derived Human Platelet Program received a great deal of media coverage in late 2012, including being awarded the December 2012 honor of being named one of the 10 Ideas that Will Shape the Yearby New Scientist Magazine,[17] unfortunately the company did not succeed in moving the concept through to clinical testing in 2013.

Nonetheless, in a November 2015 presentation by Astellas President and CEO, Yoshihiko Hatanaka, he indicated that the company will aim to develop an Ophthalmic Disease Cell Therapy Franchise based around its embryonic stem cells (ESCs) and induced pluripotent stem cell (iPS cells) technology. [18]

On June 22, 2016, RIKEN announced that it is resuming its retinal induced pluripotent stem cell (iPSC) study in partnership with Kyoto University.

2013 was the first time in which clinical research involving transplant of iPSCs into humans was initiated, led by Masayo Takahashi of the RIKEN Center for Developmental Biology (CDB)in Kobe, Japan. Dr. Takahashi and her team wereinvestigating the safety of iPSC-derived cell sheets in patients with wet-type age-related macular degeneration. Althoughthe trial was initiated in 2013 and production of iPSCs from patients began at that time, it was not until August of 2014 that the first patient, a Japanese woman, was implanted with retinal tissue generated using iPSCs derived from her own skin cells.

A team of three eye specialists, led by Yasuo Kurimoto of the Kobe City Medical Center General Hospital, implanted a 1.3 by 3.0mm sheet of iPSC-derived retinal pigment epithelium cells into the patients retina.[19]Unfortunately, the study was suspended in 2015 due to safety concerns. As the lab prepared to treat the second trial participant, Yamanakas team identified two small genetic changes in the patients iPSCs and the retinal pigment epithelium (RPE) cells derived from them. Therefore, it is major news that theRIKEN Institute will now be resuming the worlds first clinical study involving the use of iPSC-derived cells in humans.

According to the Japan Times, this attempt at the clinical studywill involve allogeneic rather than autologous iPSC-derived cells for purposes of cost and time efficiency.Specifically,the researchers will be developing retinal tissues from iPS cells supplied by Kyoto Universitys Center for iPS Cell Research and Application, an institution headed by Nobel prize winner Shinya Yamanaka. To learn about this announcement, view this article fromAsahi Shimbun, aTokyo- based newspaper.

Australian stem cell company Cynata Therapeutics (ASX:CYP) is taking a unique approach. It is creating allogeneic iPS cell derived mesenchyal stem cell (MSCs).Cynatas Cymerus technology utilizes iPSCs originating from an adult donor as the starting material for generating mesenchymoangioblasts (MCAs), and subsequently, for manufacturing clinical-gradeMSCs.

One of the key inventors of the approach is Igor Slukvin, who has released more than 70 publications about stem cell topics, including the landmark article in Cell describing the now patented Cymerus technique. Dr. Slukvins co-inventor is James Thomson, the first person to isolate an embryonic stem cell (ESC) and one of the first people to create a human-induced, pluripotent stem cell (hiPSC).

Recently, Cynata received advice from the UK Medicines and Healthcare products Regulatory Agency (MHRA) that its Phase I clinical trial application has been approved, titledAn Open-Label Phase 1 Study to Investigate the Safety and Efficacy of CYP-001 for the Treatment of Adults With Steroid-Resistant Acute Graft Versus Host Disease. It will be the worlds first clinical trial involving a therapeutic product derived from allogeneic (unrelated to the patient) induced pluripotent stem cells (iPSCs).

Participants for Cynatas upcoming Phase I clinical trial will be adults who have undergone an allogeneic haematopoietic stem cell transplant (HSCT) to treat a haematological disorder and subsequently been diagnosed with steroid-resistant Grade II-IV GvHD.The primary objective of the trial is to assess safety and tolerability, while the secondary objective is to evaluate the efficacy of two infusions of CYP-001 in adults with steroid-resistant GvHD.

There are four key advantages of Cynatas proprietary Cymerus MSC manufacturing platform, as described below.

Unlimited Quantities Cynatas Cymerus technology utilizes iPSCs originating from an adult donor as the starting material for generating mesenchymoangioblasts (MCAs), and subsequently, for manufacturing clinical-gradeMSCs. According to Cynatas Executive Chairman Stewart Washer who was recently interviewed by The Life Sciences Report, The Cymerus technology gets around the loss of potency with the unlimited iPS cellor induced pluripotent stem cellwhich is basically immortal.

Uniform Batches Because the proprietary Cymerus technology allows nearly unlimited production of MSCs from a single iPSC donor, there is batch-to-batch uniformity. Utilizing a consistent starting material allows for a standardized cell manufacturing process and a consistent cell therapy product.

Single Donor As described previously, Cynatas Cymerus technology creates iPSC-derived mesenchymoangioblasts (MCAs), which are differentiated into MSCs. Unlike other companies involved with MSC manufacturing, Cynata does not require a constant stream of new donors in order to source fresh stem cells for its cell manufacturing process, nor does it require the massive expansion of MSCs necessitated by reliance on freshly isolated donations.

Economic Manufacture at Commercial Scale (Low Cost) Finally, Cynata has achieved a cost-savings advantage through its uniqueapproach to MSCmanufacturing. Its proprietary Cymerus technology addresses a critical shortcoming in existing methods of production of MSCs for therapeutic use, which is the ability to achieve economic manufacture at commercial scale.

Footnotes [1] CellularDynamics.com (2014). About CDI. Available at: http://www.cellulardynamics.com/about/index.html. Web. 1 Apr. 2015. [2] Ibid. [3] Takahashi K, Yamanaka S (August 2006).Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell126(4): 66376. [4] 2012 Nobel Prize in Physiology or Medicine Press Release. Nobelprize.org. Nobel Media AB 2013. Web. 7 Feb 2014. Available at: http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/press.html. Web. 1 Apr. 2015. [5] Striklin, D (Jan 13, 2014). Three Companies Banking on Regenerative Medicine. Wall Street Cheat Sheet. Retrieved Feb 1, 2014 from, http://wallstcheatsheet.com/stocks/3-companies-banking-on-regenerative-medicine.html/?a=viewall. [6] Striklin, D (2014). Three Companies Banking on Regenerative Medicine. Wall Street Cheat Sheet [Online]. Available at: http://wallstcheatsheet.com/stocks/3-companies-banking-on-regenerative-medicine.html/?a=viewall. Web. 1 Apr. 2015. [7] Cellular Dynamics International (July 30, 2013). Cellular Dynamics International Announces Closing of Initial Public Offering [Press Release]. Retrieved from http://www.cellulardynamics.com/news/pr/2013_07_30.html. [8] Investors.cellulardynamics.com,. Cellular Dynamics Manufactures Cgmp HLA Superdonor Stem Cell Lines To Enable Cell Therapy With Genetic Matching (NASDAQ:ICEL). N.p., 2015. Web. 7 Mar. 2015. [9] Ibid. [10] Cellulardynamics.com,. Cellular Dynamics | Mycell Products. N.p., 2015. Web. 7 Mar. 2015. [11]Sirenko, O. et al. Multiparameter In Vitro Assessment Of Compound Effects On Cardiomyocyte Physiology Using Ipsc Cells.Journal of Biomolecular Screening18.1 (2012): 39-53. Web. 7 Mar. 2015. [12] Sciencedirect.com,. Prevention Of -Amyloid Induced Toxicity In Human Ips Cell-Derived Neurons By Inhibition Of Cyclin-Dependent Kinases And Associated Cell Cycle Events. N.p., 2015. Web. 7 Mar. 2015. [13] Sciencedirect.com,. HER2-Targeted Liposomal Doxorubicin Displays Enhanced Anti-Tumorigenic Effects Without Associated Cardiotoxicity. N.p., 2015. Web. 7 Mar. 2015. [14] Cellular Dynamics International, Inc. Fujifilm Holdings To Acquire Cellular Dynamics International, Inc.. GlobeNewswire News Room. N.p., 2015. Web. 7 Apr. 2015. [15] Ibid. [16] Advanced Cell Technologies (Feb 11, 2011). Advanced Cell and Colleagues Report Therapeutic Cells Derived From iPS Cells Display Early Aging [Press Release]. Available at: http://www.advancedcell.com/news-and-media/press-releases/advanced-cell-and-colleagues-report-therapeutic-cells-derived-from-ips-cells-display-early-aging/. [17] Advanced Cell Technology (Dec 20, 2012). New Scientist Magazine Selects ACTs Induced Pluripotent Stem (iPS) Cell-Derived Human Platelet Program As One of 10 Ideas That Will Shape The Year [Press Release]. Available at: http://articles.latimes.com/2009/mar/06/science/sci-stemcell6. Web. 9 Apr. 2015. [18] Astellas Pharma (2015). Acquisition of Ocata Therapeutics New Step Forward in Ophthalmology with Cell Therapy Approach. Available at: https://www.astellas.com/en/corporate/news/pdf/151110_2_Eg.pdf. Web. 29 Jan. 2017. [19]Cyranoski, David. Japanese Woman Is First Recipient Of Next-Generation Stem Cells. Nature (2014): n. pag. Web. 6 Mar. 2015.

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Companies Developing Induced Pluripotent Stem Cell (iPS …

Cell Science & Therapy – omicsonline.org

Index Copernicus Value: 5.12

NLMID: 101550241

The Journal of Cell Science & Therapy is an Open Access, peer-reviewed, academic journal with a wide range of fields within the discipline creates a platform for the authors to publish their comprehensive and most reliable source of information on the discoveries and current developments in the mode of original articles, review articles, case reports, short communications, etc, making them freely available through online without any restrictions or any other subscriptions to researchers worldwide.

The journal is using Editorial Manager System for quality in peer review process. Editorial Manager is an online manuscript submission, review and tracking systems. Review processing is performed by the editorial board members of Journal of Cell Science & Therapy or outside experts; at least two independent reviewers approval followed by editor approval is required for acceptance of any citable manuscript. Authors may submit manuscripts and track their progress through the system, hopefully to publication. Reviewers can download manuscripts and submit their opinions to the editor. Editors can manage the whole submission/review/revise/publish process.

Journal of Cell Science & Therapy is a peer reviewed scientific journal known for rapid dissemination of high-quality research. This Cell Science journal with highest impact factor offers an Open Access platform to the authors in academia and industry to publish their novel research. It serves the International Scientific Community with its standard research publications.

Cells are small compartments that hold the biological equipment necessary to keep an organism alive and successful. Living things may be unicellular or multicellular such as a human being. According to cell theory, cells are the fundamental unit of structure and function in all living organisms and come from preexisting cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.

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Cell Science & Therapy, Cell & Developmental Biology, Cell Biology: Research & Therapy, Cellular and Molecular Biology, Single Cell Biology, Current Opinion in Cell Biology, Cytology and Histology, Current Protocols in Stem Cell Biology, Current Stem Cell Research and Therapy, Developmental Cell, DNA and Cell Biology

The cytokines produced by expression from suitable cloning vectors containing the desired cytokine gene, can be expressed in yeast (Saccharomyces cerevisiae expression system), bacteria (Escherichia coli expression system), mammalian cells (BHK, CHO, COS, Namalwa), or insect cell systems. Cytokines are designed for demanding applications such as cell culture, differentiation studies, and functional assays mainly in the fields of immunology, neurology, and stem cell research.

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Clinical & Cellular Immunology, Advances in Molecular Diagnostics, Insights in Cell Science, Cellular and Molecular Biology, Insights in Stem Cells, International Journal of Interferon, Cytokine and Mediator Research, Journal of Interferon and Cytokine Research, Cytokine, Cytokine and Growth Factor Reviews, Lymphokine and Cytokine Research

Hematology is the investigation of blood, the blood-framing organs, and blood diseases in which the specialists deal with the diagnosis, treatment and overall management of people with blood disorders ranging from anemia to blood cancer. Some of the diseases treated by haematologists include Iron deficiency anaemia, Sickle cell anemia, Polycythemia or excess production of red blood cells, Myelofibrosis, Leukemia, hemophilia, myelodysplastic syndromes, Malignant lymphomas, Blood transfusion and bone marrow stem cell transplantation

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Immunobiology, Cytokine Biology, Hematology & Thromboembolic Diseases, Cell Signaling, Pediatric Hematology/Oncology and Immunopathology, Korean Journal of Hematology, Clinical Advances in Hematology and Oncology, Critical Reviews in Oncology/Hematology, Current Opinion in Hematology

Cell biology (cytology) is a branch of biology that studies cells their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division, death and cell function. Research in cell biology is closely related to genetics, biochemistry, molecular biology, immunology, and developmental biology.

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Cell Science & Therapy, Cell & Developmental Biology, Cellular and Molecular Biology, Cell Biology: Research & Therapy, Molecular Biology, Genes to Cells, Journal of Molecular Cell Biology, Biology of the Cell, Developmental Cell, Developmental Cell, Eukaryotic Cell, European Cells and Materials

A hair follicle is part of the skin that grows hair by packing old cells together. Attached to the follicle is a sebaceous gland, a tiny sebum-producing gland found everywhere except on the palms, lips and soles of the feet. The follicle cells that extrude hairs from just below the surface of the skin are simply too hard to bring back to life, and even preventative therapies didnt seem to be able to do much to keep them alive. But research on inducing stem cells to grow into follicle cells could change that forever.

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Cell Science & Therapy, Hair : Therapy & Transplantation, Genetic Syndromes & Gene Therapy, Insights in Stem Cells, Stem Cell Research & Therapy, Tissue Science & Engineering, Annual Review of Cell and Developmental Biology, Apoptosis : an international journal on programmed cell death, Analytical Cellular Pathology, Cellular Oncology

Mesenchymal stem cells (MSCs), the major stem cells for cell therapy. From animal models to clinical trials, MSCs have afforded promise in the treatment of numerous diseases, mainly tissue injury and immune disorders. Cell sources for MSC administration in clinical applications, and provide an overview of mechanisms that are significant in MSC-mediated therapies. Although MSCs for cell therapy have been shown to be safe and effective, there are still challenges that need to be tackled before their wide application in the clinical research field.

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Stem Cell Research & Therapy, Single Cell Biology, Cell & Developmental Biology, Insights in Cell Science, Animal Cells and Systems, Annals of the Romanian Society for Cell Biology, Annual Review of Cell and Developmental Biology, Apoptosis: an international journal on programmed cell death

Ovation Cell Therapy Hair Treatment nourishes hair and scalp with proteins and amino acids that bind and absorb into the hair shaft for hair that is noticeably thicker, stronger, and longer. The Ovation Cell Therapy is the heart of the system and is often where the system draws occasional criticism for its claims to accelerate hair growth and reduce breakage and hair loss.

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Cell Science & Therapy, Cancer Science & Therapy, Insights in Stem Cells, Stem Cell Research & Therapy Cancer Biology and Therapy, Cytotherapy, Immunotherapy, International Journal of Clinical Pharmacology Therapy and Toxicology, Japanese Journal of Cancer and Chemotherapy

The external effects of degenerative processes inside the body which manifest especially in the face, hands, dcollet, and by hair loss are also psychically stressful. There are promising therapeutic approaches with stem cells and growth factors for both skin regeneration and hair growth regeneration. To dispense with hair transplants and surgical procedures such as facelifts and eyelid correction, in which the skin is pulled back and the excess tissue is excised. To treat the root cause and restore lost volume in a tissue-conserving, natural manner and regenerate both the subcutaneous tissue and the skin.

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Single Cell Biology, Genetic Syndromes & Gene Therapy, Cell Science & Therapy, Cell Biology: Research & Therapy, Journal of immunotherapy, Photo-dermatology, Case Reports in Dermatology, Current Stem Cell Research and Therapy, Dermatologic Therapy

Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy.

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Cell Science & Therapy, Insights in Cell Science, Cellular and Molecular Biology, Cell Biology: Research & Therapy, Hematology/Oncology and Stem Cell Therapy, Journal of Cosmetic and Laser Therapy, Cancer Biology and Therapy, Cancer Gene Therapy, Cytotherapy

Rejuvenation and regeneration are two key processes that define cell therapy. Cellular Therapy is a form of non-toxic, holistic medicine in which the entire organism is being treated. Cellular Therapies are an integral part of complimentary treatment regimens. They are extremely versatile and can be used for a wide range of disorders.

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Cell & Developmental Biology, Archives in Cancer Research,Cancer Clinical Trials, Cancer Science & Therapy, Cancer Biology and Therapy, Cancer Gene Therapy, Cytotherapy, Journal of Cancer Science and Therapy, Stem Cell Research and Therapy

Dendritic cells (DCs) cells are the most potent antigen-producing cells, represent unique antigen-producing cells capable of sensitizing T cells to both new and recall antigens. Dendritic Cell Vaccines, or Dendritic cell therapy, is another Alternative Cancer Therapy or newly emerging and potent form of immune therapy used to treat cancer.

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Clinical & Experimental Neuroimmunology, Immunochemistry & Immunopathology: Open Access, Clinical & Cellular Immunology, Immunooncology, Dendrobiology, Genes and Cancer, International Journal of Cancer, Journal of Cancer Science and Therapy, Molecular Cancer Research, Molecular Cancer Therapeutics

The cells are most commonly immune-derived, with the goal of transferring immune functionality and characteristics along with the cells. Transferring autologous cells minimizes GVHD issues. The adaptive transfer of autologous tumor infiltrating lymphocytes (TIL) or genetically re-directed peripheral blood mononuclear cells has been used to treat patients with advanced solid tumors, including melanoma and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies. As of 2015 the technique had expanded to treat cervical cancer, lymphoma, leukemia, bile duct cancer and neuroblastoma.

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Cell Signaling, Cellular & Molecular Pathology, Cell Biology: Research & Therapy, Stem Cell Research & Therapy, Antiviral Chemistry and Chemotherapy, Cancer Biotherapy and Radiopharmaceuticals, Cancer Biology and Therapy, Cytotherapy, Japanese Journal of Cancer and Chemotherapy, Oncology and Stem Cell Therapy

The ability to convert one cell type into another has caused great excitement in the stem cell field. iPS Reprogramming and transdifferentiation are the two approaches which makes cells in to another type of cells. In iPS procedure, it make possible to convert essentially any cell type in the body back into pluripotent stem cells that are almost identical to embryonic stem cells. And another approach uses transcription factors to convert a given cell type directly into another specialized cell type, without first forcing the cells to go back to a pluripotent state.

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Autologous stem cell transplants are done using peripheral blood stem cell transplantation (PBSCT). With PBSCT, the stem cells are taken from blood. The growth factor G-CSF may be used to stimulate the growth of new stem cells so they spill over into the blood.

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Advance Cell & Gene Thearpy practical, experienced guidance in development, GMP/GTP manufacturing, and regulatory compliance, as well as comprehensive scientific and technical strategic analysis of business opportunities in cell therapy, gene therapy and tissue therapies.

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Immunotherapy involves engineering patients own immune cells to recognize and attack their tumors. And although this approach, called adoptive cell transfer (ACT), has been restricted to small clinical trials so far, treatments using these engineered immune cells have generated some remarkable responses in patients with advanced cancer. .Adoptive T cell therapy for cancer is a form of transfusion therapy consisting of the infusion of various mature T cell subsets with the goal of eliminating a tumor and preventing its recurrence.

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Clinical & Cellular Immunology, Immunooncology, Molecular Immunology, Advances in Cancer Prevention, Cytotherapy, Journal of Acquired Immune Deficiency Syndromes, Advances in Neuroimmune Biology, Cancer Biology and Therapy, Cancer Immunology, Immunotherapy

Commercialization of the first cell-based therapeutics, including cartilage repair products; tissue-engineered skin; and the first personalized, cellular immunotherapy for cancer. Production, storage, and delivery of living cell-based pharmaceuticals presents several unique challenges. Novel, innovative technologies and strategies will be required to bring cell therapies to commercial success.

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Bioprocessing & Biotechniques, Cytology & Histology, Cell Biology: Research & Therapy , Molecular Biology, BioProcess International, Biotechnology and Bioprocess Engineering, Food and Bioprocess Technology, Industrial Bioprocessing

Cellular therapy products include cellular immunotherapies, and other types of both autologous and allogeneic cells for certain therapeutic indications, including adult and embryonic stem cells. Human gene therapy refers to products that introduce genetic material into a persons DNA to replace faulty or missing genetic material, thus treating a disease or abnormal medical condition.

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Pharmacognosy & Natural Products, Natural Products Chemistry & Research, Stem Cell Research & Therapy, Cell Science & Therapy, Surgical Products, International Journal of Applied Research in Natural Products, Molecular Diagnosis and Therapy, Molecular Therapy, Molecular Therapy – Nucleic Acids

Journal of Cell Science and Therapy is associated with our international conference “6th World Congrss on Cell & Stem Cell Research” during Feb 29- March 2, 2016 Philadelphia, USA with a theme “Novel Therapies in Cell Science and Stem Cell Research. Stem Cell Therapy-2016 will encompass recent researches and findings in stem cell technologies, stem cell therapies and transplantations, current understanding of cell plasticity in cancer and other advancements in stem cell research and cell science.

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Cell Science & Therapy – omicsonline.org

Induced pluripotent stem-cell therapy – Wikipedia

In 2006, Shinya Yamanaka of Kyoto University in Japan was the first to disprove the previous notion that reversible cell differentiation of mammals was impossible. He reprogrammed a fully differentiated mouse cell into a pluripotent stem cell by introducing four genes, Oct-4, SOX2, KLF4, and Myc, into the mouse fibroblast through gene-carrying viruses. With this method, he and his coworkers created induced pluripotent stem cells (iPS cells), the key component in this experiment.[1] Scientists have been able to conduct experiments that show the ability of iPS cells to treat and even cure diseases. In this experiment, tests were run on mice with inherited sickle-cell anemia. Skin cells were turned into cells containing genes that transformed the cells into iPS cells. These replaced the diseased sickled cells, curing the test mice. The reprogramming of the pluripotent stem cells in mice was successfully duplicated with human pluripotent stem cells within about a year of the experiment on the mice.[citation needed]

Sickle-cell anemia is a disease in which the body produces abnormally shaped red blood cells. Red blood cells are flexible and round, moving easily through the blood vessels. Infected cells are shaped like a crescent or sickle (the namesake of the disease). As a result of this disorder the hemoglobin protein in red blood cells is faulty. Normal hemoglobin bonds to oxygen, then releases it into cells that need it. The blood cell retains its original form and is cycled back to the lungs and re-oxygenated.

Sickle cell hemoglobin, however, after giving up oxygen, cling together and make the red blood cell stiff. The sickle shape also makes it difficult for the red blood cell to navigate arteries and causes blockages.[2] This can cause intense pain and organ damage. The sickled red blood cells are fragile and prone to rupture. When the number of red blood cells decreases from rupture (hemolysis), anemia is the result. Sickle cells die in 1020 days as opposed to the traditional 120-day lifespan of a normal red blood cell.

Sickle cell anemia is inherited as an autosomal (meaning that the gene is not linked to a sex chromosome) recessive condition.[2] This means that the gene can be passed on from a carrier to his or her children. In order for sickle cell anemia to affect a person, the gene must be inherited from both the mother and the father, so that the child has two recessive sickle cell genes (a homozygous inheritance). People who inherit one sickle cell gene from one parent and one normal gene from the other parent, i.e. heterozygous patients, have a condition called sickle cell trait. Their bodies make both sickle hemoglobin and normal hemoglobin. They may pass the trait on to their children.

The effects of sickle-cell anemia vary from person to person. People who have the disease suffer from varying degrees of chronic pain and fatigue. With proper care and treatment, the quality of health of most patients will improve. Doctors have learned a great deal about sickle cell anemia since its discovery in 1979. They know its causes, its effects on the body, and possible treatments for complications. Sickle cell anemia has no widely available cure. A bone marrow transplant is the only treatment method currently recognized to be able to cure the disease, though it does not work for every patient. Finding a donor is difficult and the procedure could potentially do more harm than good. Treatments for sickle cell anemia are generally aimed at avoiding crises, relieving symptoms, and preventing complications. Such treatments may include medications, blood transfusions, and supplemental oxygen.

During the first step of the experiment, skin cells (also known as fibroblasts) were collected from infected test mice and put in a culture. The fibroblasts were reprogrammed by infecting them with retroviruses that contained genes common to embryonic stem cells. These genes were the same four used by Yamanaka (Oct-4, SOX2, KLF4, and Myc) in his earlier study. The investigators were trying to produce cells with the potential to differentiate into any type of cell needed (i.e. pluripotent stem cells). As the experiment continued, the fibroblasts multiplied into identical copies of iPS cells. The cells were then treated to form the mutation needed to reverse the anemia in the mice. This was accomplished by restructuring the DNA containing the defective globin gene into DNA with the normal gene through the process of homologous recombination. The iPS cells then differentiated into blood stem cells, or hematopoietic stem cells. The hematopoietic cells were injected back into the infected mice, where they proliferate and differentiate into normal blood cells, curing the mice of the disease.[3][4][verification needed]

To determine whether the mice were cured from the disease, the scientists checked for the usual symptoms of sickle cell disease. They examined the blood for mean corpuscular volume (MCV) and red cell distribution width (RDW) and urine concentration defects. They also checked for sickled red blood cells. They examined the DNA through gel electrophoresis, checking for bands that display an allele that causes sickling. Compared to the untreated mice with the disease, which they used as a control, “the treated animals had marked increases in RBC counts, healthy hemoglobin, and packed cell volume levels”.[5]

Researchers examined “the urine concentration defect, which results from RBC sickling in renal tubules and consequent reduction in renal medullary blood flow, and the general deteriorated systemic condition reflected by lower body weight and increased breathing.”[5] They were able to see that these parts of the body of the mice had healed or improved. This indicated that “all hematological and systemic parameters of sickle cell anemia improved substantially and were comparable to those in control mice.”[5] They cannot say if this will work in humans because a safe way to inject the genes for the induced pluripotent cells is still needed.[citation needed]

The reprogramming of the induced pluripotent stem cells in mice was successfully duplicated in humans within a year of the successful experiment on the mice. This reprogramming was done in several labs and it was shown that the iPS cells in humans were almost identical to original embryonic stem cells (ES cells) that are responsible for the creation of all structures in a fetus.[1] An important feature of iPS cells is that they can be generated with cells taken from an adult, which would circumvent many of the ethical problems associated with working with ES cells. These iPS cells also have potential in creating and examining new disease models and developing more efficient drug treatments.[6] Another feature of these cells is that they provide researchers with a human cell sample, as opposed to simply using an animal with similar DNA, for drug testing.

One major problem with iPS cells is the way in which the cells are reprogrammed. Using gene-carrying viruses has the potential to cause iPS cells to develop into cancerous cells.[1] Also, an implant made using undifferentiated iPS cells, could cause a teratoma to form. Any implant that is generated from using these iPS cells would only be viable for transplant into the original subject that the cells were taken from. In order for these iPS cells to become viable in therapeutic use, there are still many steps that must be taken.[5][7]

In the future, researchers hope that induced pluripotent cells may be used to treat other diseases. Pluripotency is a crucial part of disease treatment because iPS cells are capable of differentiation in a way that is very similar to embryonic stem cells which can grow into fully differentiated tissues. iPS cells also demonstrate high telomerase activity and express human telomerase reverse transcriptase, a necessary component in the telomerase protein complex. Also, iPS cells expressed cell surface antigenic markers expressed on ES cells. Also, doubling time and mitotic activity are cornerstones of ES cells, as stem cells must self-renew as part of their definition. As said, iPS cells are morphologically similar to embryonic stem cells. Each cell has a round shape, a large nucleolus and a small amount of cytoplasm. One day, the process may be used in practical settings to provide a fundamental way of regeneration.

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Induced pluripotent stem-cell therapy – Wikipedia

Cellular Therapy Training Course – ISCT

The Inaugural ISCT-ASBMT Cell Therapy Training Course One Scholars Experience

Beth Sage MBBS, PhD UCL Respiratory University College London, London, UK

Having been lucky enough to be selected as an international scholar for the inaugural Cell Therapy Training Course I was looking forward to leaving behind the rather disappointing British summer and heading towards the much warmer Houston fall. Having googled my destination and accommodation I boarded the plane with great excitement and hopes of enjoying the Texan heat whilst exploring the cosmopolitan offerings of Americas fourth largest city oh and learning something about cell therapy!

The course, chaired by Dave DiGiusto and John Barrett, was the first of its kind, a joint enterprise between the ISCT and the American Society of Blood and Marrow Transplantation. Following a competitive selection procedure 12 scholars from all over the world were invited to attend a 5 day intensive workshop with the primary objective of giving junior cell therapy researchers an insight into the process of taking their research project from bench to bedside, including processing, clinical trial design, regulatory requirements, commercialization and ethical research. Alongside didactic lecture based teaching there were tours of good manufacturing practice (GMP) facilities, both academic and commercial, and most anticipated by the scholars was the opportunity to participate in small group discussions, led by experts in the field, dissecting and improving the individual cell therapy projects.

To break the ice on the light first day each scholar gave a short presentation on their project. It was immediately clear that there is a great breadth of exciting, novel cell therapy projects under investigation throughout the world, from the use of modified T-cells in hematological malignancies to the development of a tissue-engineered oesophagus using amniotic fluid stem cells. Projects ranged from early pre-clinical to those embarking on a first in man clinical trial and every stage in between, making the session interesting and varied. Having fought the jet-lag, the session ended with an ice-breaker drinks and dinner before retiring to prepare for the days ahead.

Over the next few days we were exposed to a wealth of information with detailed talks on pre-clinical development of different cell therapies from CD34 cells to mesenchymal stromal cells, quality systems development and one of the most useful from my personal perspective, manufacturing and release testing of different products. We were able to visit different manufacturing facilities and to understand the processes involved in the production of a clinical grade therapy. It made us challenge the protocols we were developing in the lab as we gained an insight into how it would scale up into a commercially viable process a 26 day culture process of autologous cells requiring purification and multiple cytokine stimulations is significantly more challenging (and expensive) than allogeneic cells cultured for 14 days with no manipulation and simple media exchanges.

Once the process development sessions were complete, we switched gears to look at how to conduct cell therapy clinical trials, covering issues of producing products including normal donors that are used to treat multiple recipients, the challenges of pooling donor cells, how to run multicenter studies and most importantly (although I can say almost universally never thought about by the scholars) how to deal with a regulatory body audit. This really was a really informative session that opened our eyes to the challenges and complexities of working in the field of cell therapy trials.

Just when we were beginning to feel that our jobs over the next few years would be focused on clinical trial design, process validation and filling in an endless paper chain of regulatory documents we were brought back to where we all started the excitement of the translational science. This was, for me, a really interesting session on the importance of correlative studies not only to assess clinical trial performance but to provide mechanistic insights into the behavior of cells when delivered to patients with disease. As scientists we can design and perform many experiments to predict how manipulated cells will behave but the most important data of all comes from the patients themselves. For me, the importance of testing a novel therapy is not just to see if it works but how it works and, just as importantly, if it doesnt – why.

To end to course, our wise leaders Dave DiGiusto and John Barrett decided to test whether we had been listening, and each scholar had to deliver a detailed presentation on how their project had developed during the course. Each scholar had to address the potential pitfalls and specific challenges they faced in moving towards the clinic. Whilst many of us stayed up into the small hours worrying about aspects we were previously oblivious to, undoubtedly we found this one of the most rewarding moments. Despite the potential difficulties we were now aware of, we also felt better placed to solve them and could see a clearer path ahead.

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Cellular Therapy Training Course – ISCT

Stem Cell FAQ

Some of the promise of stem cell therapy has been realized. A prime example is bone marrow transplantation. Even here, however, manyproblems remain to be solved.

Challenges facing stem cell therapy include the following:

Adult stem cells Tissue-specific stem cells in adult individuals tend to be rare. Furthermore, while they can regenerate themselves in an animal or person they are generally very difficult to grow and to expand in the laboratory. Because of this, it is difficult to obtain sufficient numbers of many adult stem cell types for study and clinical use. Hematopoietic or blood-forming stem cells in the bone marrow, for example, only make up one in a hundred thousand cells of the bone marrow. They can be isolated, but can only be expanded a very limited amount in the laboratory. Fortunately, large numbers of whole bone marrow cells can be isolated and administered for the treatment for a variety of diseases of the blood. Skin stem cells can be expanded however, and are used to treat burns. For other types of stem cells, such as mesenchymal stem cells, some success has been achieved in expanding the cellsin vitro, but application in animals has been difficult. One major problem is the mode of administration. Bone marrow cells can be infused in the blood stream, and will find their way to the bone marrow. For other stem cells, such as muscle stem cells, mesenchymal stem cells and neural stem cells, the route of administration in humans is more problematic. It is believed, however, that once healthy stem cells find their niche, they will start repairing the tissue. In another approach, attempts are made to differentiate stem cells into functional tissue, which is then transplanted. A final problem is rejection. If stem cells from the patients are used, rejection by the immune system is not a problem. However, with donor stem cells, the immune system of the recipient will reject the cells, unless the immune system is suppressed by drugs. In the case of bone marrow transplantation, another problem arises. The bone marrow contains immune cells from the donor. These will attack the tissues of the recipient, causing the sometimes deadly graft-versus-host disease.

Pluripotent stem cells All embryonic stem cell lines are derived from very early stage embryos, and will therefore be genetically different from any patient. Hence, immune rejection will be major issue. For this reason, iPS cells, which are generated from the cells of the patient through a process of reprogramming, are a major breakthrough, since these will not be rejected. A problem however is that many iPS cell lines are generated by insertion of genes using viruses, carrying the risk of transformation into cancer cells. Furthermore, undifferentiated embryonic stem cells or iPS cells form tumors when transplanted into mice. Therefore, cells derived from embryonic stem cells or iPS cells have to be devoid of the original stem cells to avoid tumor formation. This is a major safety concern.

A second major challenge is differentiation of pluripotent cells into cells or tissues that are functional in an adult patient and that meet the standards that are required for ‘transplantation grade’ tissues and cells.

A major advantage of pluripotent cells is that they can be grown and expanded indefinitely in the laboratory. Therefore, in contrast to adult stem cells, cell number will be less of a limiting factor. Another advantage is that given their very broad potential, several cell types that are present in an organ might be generated. Sophisticated tissue engineering approaches are therefore being developed to reconstruct organs in the lab.

While results from animal models are promising, the research on stem cells and their applications to treat various human diseases is still at a preliminary stage. As with any medical treatment, a rigorous research and testing process must be followed to ensure long-term efficacy and safety.

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Stem Cell FAQ

Recent Advances in Hematopoietic Stem Cell Gene Therapy …

1. Introduction

Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indications are restricted because of critical complications including regimen-related toxicities involving conditioning, infection, and graft-versus-host disease.

Studies in recent decades have shown that HSCT can have a long-term effect in the treatment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined immunodeficiency (SCID-X1) resulted in tumorigenesis [4]. In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.

However, as technological advances have increased the safety and efficiency of introducing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various hereditary diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress [8].

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+CD38-c-Mpl- population, CD34+CD38-c-Mpl+ cells show significantly better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-renewal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A signal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC re-entry into the cell cycle, transforming growth factor- (TGF-) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs. Stem cell factor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor – via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc. San Diego, CA, U.S.A. http://www.biolegend.com/cell_markers)

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromosome. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentiation-specific or non-specific promoters or enhancers during the vector design process.

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activity, are widely used for inserting genes in host chromosomes. Although adeno-associated virus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called simply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a gag/pol gene that codes for viral structure proteins, protease and reverse transcriptase, an env gene that codes for the envelope glycoprotein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the gag/pol gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collecting the culture solution.

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG islands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5 LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vector, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disassembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although various combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one advantage of lentiviral vectors in HSC gene therapy.

Both lentiviruses and gammaretroviruses have gag, pol, and env genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (vif, vpr, vpu, nef) and regulatory genes (tat, rev). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, allowing the viral genome to be inserted into the host cell chromosome.

HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B). The viral long terminal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the gag and pol genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human -globin gene. As the transcripts of the gag and pol genes contain cis-repressive sequences, they are expressed only if rev promotes their nuclear export by binding to the RRE. All tat and rev exons have been deleted, and the viral sequences upstream of the gag gene have been replaced. The rev plasmid expresses rev cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5 LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on tat. The 3 LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the integrated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivating (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with modifications from [26]).

Although first-generation lentiviral vectors included modification genes, they were removed in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifications included the deletion of tat, use of multiple vector plasmids, and introduction of self-inactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the packaging plasmid, rev plasmid, SIN vector plasmid and envelope plasmid. To prevent production of wild type HIV-1, tat, a regulatory gene indispensable to viral reproduction was deleted, and the rev gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of tat, it was replaced with the cytomegalovirus (CMV) promoter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replacing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary genes with the integrated gene (Fig. 3) [27].

Mechanism of gene activation induced by vector insertion. The genomic integration site of an MLV-based retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a self-inactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modification from [27]).

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vectors displaying early-acting cytokines such as TPO and SCF. This vector can promote survival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].

Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient targeting of hematopoietic stem cells. Blood. 2005;106(10):3333)

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the development of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular proto-oncogene and mediates transformation. This mechanism does not occur in replication-incompetent vectors. Second, the provirus 3 LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5 LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector [30].

Retroviral mechanisms of oncogenesis. The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes respectively (Reproduced from [30]).

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-Gpseudotyped HIV vectors have an integration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integration sites.

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than retroviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene undergoes differentiation.

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and metabolic diseases. Most are congenital or hereditary diseases. The characteristic clinical features and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.

Clinical applications of hematopoietic stem cell gene therapy.

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two -globin and two -globin chains combined with a heme group. -thalassemia is an autosomal hemoglobin disorder caused by decreased -globin chain synthesis. Although individuals with -thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, -thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfusion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector containing a functional -globin gene has been performed in an HbE/ -thalassemia (E/ 0) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months [33].

The human -globin locus is located in a large 70kb area which also contains some -like globulin genes (, G, A, , ). Gene switching takes place according to the development stage, and the -globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5 side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5 side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

The structure of the lentiviral SIN vector used in gene therapy for -thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal () of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken -globin insulators) were inserted in the U3 region of the HIV 3 LTR. Furthermore, the amino acid at the 87th position of -globin was changed from threonine to glutamine. This altered -globin can be distinguished from normal adult -globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and +-thalassemia patients [33].

Diagram of the human -globin gene in a lentiviral vector. HIV LTR, human immunodeficiency type-1 virus long terminal repeat; +, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive element; p, human -globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color modification from [33])

A clinical study using this vector was performed in two -thalassemia patients. As with autologous bone marrow transplantation, some of the patients marrow cells were cryopreserved as a backup. The lentiviral vector particles containing a functional -globin were introduced into the remaining cells. After the transfected cells were cultured for one week ex vivo, some were also cryopreserved. The patients were conditioned with intravenous busulfan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before transplantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term -globin production; one-third of the patients hemoglobin was produced by the genetically modified cells [33].

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the HMGA2 gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was expressive production of a truncated form of the HMGA2 protein. Since truncated or overexpressed HMGA2 is observed with some blood cancers and non-malignant expansions of blood cells, caution is recommended with this therapy [34].

Gene-therapy procedure for patient with b-thalassemia. a. Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with -thalassemia and maintained them in culture. b, Lentiviral-vector particles containing a functional -globin gene were then introduced into the cells and allowed them to expand further in culture. c. To eradicate the patients remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemotherapy. d. The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two hereditary persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic levels of A-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated -globin gene addition and genetic reactivation of endogenous -globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with -globin deficiency [36]. In addition, a trial of -globin induction with -globin production using mithramycin, an inducer of -globin expression, to remove excess -globin proteins in -thalassemic erythroid progenitor cells was reported [37].

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approximately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the ten-year probability of survival after transplant from an Human leukocyte antigen (HLA) -identical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN) have been cloned. Mutations in FANCB are associated with an X-linked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, FANCA gene abnormalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during ex vivo culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the FANCA and FANCC genes [20, 42-44]. In a phase I trial of FANCA gene therapy, gene transfer was performed with patient bone marrow-derived CD34+ cells and the MSCV retroviral vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi anemia patients due to possible bone marrow insufficiency, but in this study, sufficient target CD34+ cells were obtained from most patients. Two patients had FANCA-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient improvements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clinical study of FANCC gene therapy using a retroviral vector involved four patients. Although functional FANCC gene expression was observed in peripheral blood and bone marrow cells, the results were transient [43].

Engraftment efficiency of FANCA-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakaryocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleukin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with FANCA abnormalities with short transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-L-cysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells mediates efficient phenotypic correction of hematopoietic progenitor cells and CD34- mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In Fancg -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs in vitro [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for FANCC gene transfer in mice was able to successfully introduce the FANCC gene to HSCs. This result provides evidence that targeting the HSCs directly in their native environment enables efficient and long-term correction of bone marrow defects in Fanconi anemia [47].

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of FANCA gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of FANCA. The PGK-FANCA lentiviral vectors with either a wild-type woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3 region have higher levels of FANCA gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and efficiency for Fanconi anemia gene therapy [48].

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a FANCA gene abnormality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed in patients with hematologic disorders [49].

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemophilia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with hemophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually produced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibroblasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vector or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein expression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, partial replacement of the mutated liver cells by healthy cells in hemophilia A mice was challenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia [55].

Although HSCT has been widely performed as curative treatment for primary immunodeficiencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, frequent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lymphocytes continued for two years after the cessation of treatment [1], gene therapy attracted attention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered [56].

SCID-X1 is an X-linked disease caused by deficiency of the common (c) chain in the IL-2 receptor. Because the c chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be achieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. After gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as LIM domain only 2 (LMO2), Bmi1, cyclin D2 (CCND2) are near the gene insertion region, there was a high frequency of T-cell leukemia after treatment. Furthermore, in the patients who developed leukemia, additional chromosomal changes, including activating mutations of Notch1, changes in the T cell receptor region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (CDKN2A) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 positive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus influences the target cells dormant capacity for survival, engraftment, and proliferation.

Although continuous T cell production was founded in many cases, there was little reconstruction of myeloid cells and B cells, and some patients required continuous immunoglobulin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after c chain gene therapy. There is decreased NK cell reconstruction without conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after allergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children [60].

To study whether activation of genes near the region of gene insertion or inserted c chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the human c chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- c chain gene) was performed in mice. When the CD2- c chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in c chain-gene deficient mice. This study demonstrated that when the c c chain gene is expressed externally, SCID-X1 may be treated safely [61].

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or c chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive disease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetriphosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined immunodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycolmodified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase peripheral T cell counts was attempted during the early stages of gene therapy. Although adverse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the ADA gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was discontinued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Epstein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-induced leukemia [25].

As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as LMO2, or proto-oncogenes [66]. In clinical studies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, including heart, skeletal muscle, and kidney [71].

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in gp91phox on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, leading to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis reduces the incidence of infections, the overall annual mortality rate remains high (2%5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flare-ups at infected sites [56].

In the initial trials of CGD gene therapy without any conditioning therapy, p47phox or gp91phox gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte colony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74].

In a German study where gp91phox was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leukocytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as myelodysplastic syndrome 1-ecotropic virus integration site 1 (MDS1/EVI1), PR domain containing protein 16 (PRDM16), SET binding protein 1 (SETBP1). Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to EVI1 activation. One died of severe sepsis 27 months after gene therapy. Although the gene-inserted cells remained expressed in this patient, methylation of the CpG site in the LTR of the viral vector was observed and the expression of the inserted gp91phox gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although gp91phox gene expression was decreased, the activation of EVI1 near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene insertion site will be increased, there remains a concern about tumorigenesis with peripheral stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lentiviral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were studied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce sequence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of gp91phox was targeted into one allele of the “safe harbor” AAVS1 locus in iPSs [79].

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WAS gene result in a wide spectrum of clinical manifestations ranging from relatively mild X-linked thrombocytopenia to the classic WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous in vivo reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are sometimes seen [81].

In one preclinical study introducing the WAS gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigenesis is necessary, as with SCID-X1 and CGD [84, 85].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].

Since a study using human embryonic stem cells (hESCs) and WAS-promoterdriven lentiviral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expression in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs [91].

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-haplo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is successful in most cases.

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of JAK3 into the patients bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was observed for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigenesis remains a concern with JAK gene therapy [92].

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by lethal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human PNP resulted in human PNP expression, improved thymocyte maturation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of PNP-transduced cells and the percentage of engrafted cells decreased [94].

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte integrin CD11/CD18 heterodimer due to mutations in the CD18 gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious bacterial infections immediately after birth.

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the CD18 gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. In vivo animal experiments using a PGK or an elongation factor 1 promoter did not lead to symptom improvement [95-97], but improvement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral vector in one animal study [98].

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumulation into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is divided into seven types: type I (-L-iduronidase deficiency, Hurler syndrome, Sheie syndrome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, -N-acetylglucosaminidase deficiency, -glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase deficiency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syndrome), type VII (-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lysosomes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retardation, varies with MPS type.

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major challenges in the treatment of MPS. Since replaced enzymes easily cannot pass the blood-brain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for example, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, although the underlying mechanism is unknown. There are also trials where AAV vectors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue [99].

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts [101].

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or without mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, infantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is considered in cases with little improvement with enzyme replacement therapy [103].

For Gaucher disease without CNS symptoms, a animal model using an AAV vector to produce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher -glucocerebrosidase activity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelination of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (ABCD1) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be obtained with HSCT [107].

One study has reported the introduction of wild-type ABCD1 using a lentiviral vector into peripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 714% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

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Recent Advances in Hematopoietic Stem Cell Gene Therapy …

Steps Toward Safe Cell Therapy Using Induced Pluripotent …

Hideyuki Okano

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Masaya Nakamura

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Kenji Yoshida

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Yohei Okada

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Osahiko Tsuji

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Satoshi Nori

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Eiji Ikeda

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Shinya Yamanaka

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

Kyoko Miura

From the Departments of Physiology (H.O., K.Y., Y.O., O.T., S.N., K.M.) and Orthopedic Surgery (M.N., O.T., S.N.) and Kanrinmaru Project (K.Y., Y.O.), School of Medicine, Keio University, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan (E.I.); Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan (S.Y.); and Genomic Science Laboratory, Dainippon Sumitomo Pharma, Osaka, Japan (K.Y.).

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Steps Toward Safe Cell Therapy Using Induced Pluripotent …

[Retinal Cell Therapy Using iPS Cells]. – ncbi.nlm.nih.gov

Progress in basic research, starting with the work on neural stem cells in the middle 1990’s to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author’s group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient’s own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment is now being investigated in preclinical research. Among the mixture of rod and cone photoreceptors in the donor cells, the percentage of cone photoreceptors is still low. Donor cells with more. cone photoreceptors will be needed. If that will work well, photoreceptor transplantation will be the first example of neural network reconstruction in the central nervous system. These efforts will reach to variety of retinal cell transplantations in the future.

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[Retinal Cell Therapy Using iPS Cells]. – ncbi.nlm.nih.gov

[Induced Pluripotent Stem (iPS) Cell-based Cell Therapy …

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the dystrophin gene. There is currently no effective treatment for DMD. Muscle satellite cells are tissue-specific stem cells found in the skeletal muscle; these cells play a central role in postnatal muscle growth and regeneration, and are, therefore, a potential source for stem cell therapy for DMD. However, transplantation of satellite cell-derived myoblasts has not yet been successful in humans. Patient-specific induced pluripotent stem (iPS) cells are expected to be a source for autologous cell transplantation therapy for DMD, because iPS cells can proliferate vigorously in vitro and can differentiate into multiple cell lineages both in vitro and in vivo. Here, we discuss the strategies to generate muscle stem cells from iPS cells. So far, the most promising method for generating muscle stem cells from iPS cells is the conditional overexpression of Pax3 or Pax7 in the differentiating mouse embryoid bodies. However, induction methods for human iPS cells have not yet been developed. Thus, iPS cells are expected to serve as an in vitro disease model system, which will enable us to determine the pathology of muscle diseases and develop pharmaceutical treatments.

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[Induced Pluripotent Stem (iPS) Cell-based Cell Therapy …

Stem Cells Therapy IPS Cell Therapy IPS Cell Therapy

Editor-in-Chief Marek Malecki, MD PhD President Genetic and Biomolecular Engineering PBMEF, San Francisco, USA E-mail: [emailprotected]

Marek Malecki MD PhD is President of Phoenix Biomolecular Engineering Foundation, Chief Executive Officer of the Center for Molecular Medicine, and Visiting Professor at the University of Wisconsin. He earned MD degree at the Medical Academy, Poznan followed by Residency/Fellowship in Molecular Medicine in Rigshospitalet, Copenhagen. He earned PhD at the National Academy of Sciences, Warsaw followed by the postdoctoral fellowships in molecular biology at the Austrian Academy of Sciences, Karolinska Institutet, Stockholm, Salzburg and Vienna, ETH, Zurich, Utrecht University Medical School, Utrecht, Cancer Center, Vienna, Cancer Center, Amsterdam, Biozentrum, Basel. Dr Malecki developed a novel technology to identify and isolate single living pluripotent stem cells followed by their clonal expansion and molecular profiling including sequencing their proteomes, transcriptomes, and genomes. This technology serves also for reprogramming the stem cells for their use as the vectors in gene therapy of cancer. The technology, protected by the US and WIPO, is currently streamlined to clinical trials. He is the first author on the peer-reviewed articles. He is often an invited speaker and courses faculty at the international professional conferences. Dr Malecki is Editor in Chief of the Journal of Genetic Syndromes and Gene Therapy and Member of the Editorial Boards for many high-impact professional journals. He is the member of the American Medical Association, American Association of Human Genetics, American Antibody Society, Southern California Biotechnology Council, and Rho Chi Honor Society for Excellence in Teaching and as the Faculty Role Model.

cancers of ovaries, cancers of testes, cancer stem cells (CSC), circulating tumor cells (CTCs), genetic disorders, iatrogenic genetic mutations, gene therapy, targeted gene delivery, fertility sparing therapy, biobanking, in vitro fertilization.

Evan Yale Snyder, MD, PhD Professor Director, Program in Stem Cell & Regenerative Biology and Stem Cell Research Center Sanford-Burnham Medical Research Institute (SBMRI) California, USA

Evan Y. Snyder earned his M.D. and Ph.D. from the University of Pennsylvania. He completed residencies (including serving as Chief Resident) in pediatrics and neurology as well as a clinical fellowship in neonataology at Childrens Hospital-Boston, Harvard Medical School. He became a faculty physician in the Department of Pediatrics, Children & middots Hospital-Boston and while serving as a research fellow in the Department of Genetics, Harvard Medical School. He established a lab at Children & middots Hospital-Boston in 1992. In 2003, Dr. Snyder was recruited to the Burnham Institute for Medical Research as Professor and Director of the Program in Stem Cell & Regenerative Biology. He then inaugurated the Stem Cell Research Center and initiated the Southern California Stem Cell Consortium. He serves on multiple editorial boards, has published extensively in the stem cell literature, holds multiple patents in the stem cell space, has received numerous honors and lecturers widely internationally.

Fundamental stem cell biology Developmental neuroscience Neural transplantation Developmental biology Cellular (in vitro) and animal models of disease Differentiation of pluripotent and multipotent stem cells Neurodegenerative diseases Neural injury and repair Ethics and public policy Science education.

Fazlul Hoque Sarkar, PhD Distinguished Professor Departments of Pathology and Oncology Karmanos Cancer Institute Wayne State University School of Medicine Detroit, USA Read Interview session with Fazlul Hoque Sarkar

Fazlul H. Sarkar, Ph.D. is a Professor at Karmanos Cancer Center, Wayne State University with a track-record of cancer research for over 32 years. He received his Ph.D. degree in biochemistry and completed his post-doctoral training in molecular biology at Memorial Sloan Kettering Cancer Center in New York. His work has led to the discovery of the role of chemopreventive agents in sensitization of cancer cells (reversal of drug-resistance) to conventional therapeutics (chemo-radio-therapy). He has published over 400 original scientific articles in peer-reviewed journals, review articles and book chapters. He is currently a Senior Editor of the journal: Molecular Cancer Therapeutics and member of the editorial board of many scientific journals.

His research is focused on understanding the role of a master transcription factor, NF-B, and further directed toward elucidating the molecular mechanisms of action of natural agents and synthetic small molecules for cancer prevention and therapy.

LuZhe Sun, PhD Professor Department of Cellular & Structural Biology University of Texas Health Science Center San Antonio, USA

LuZhe Sun is Dielmann Endowed Chair in Oncology, Professor of Cellular & Structural Biology, University of Health Science Center at San Antonio. Associate Director for Translational Research, Cancer Treatment and Research Center, University of Health Science Center at San Antonio. He received Ph.D. in Physiology in 1990 from Rutgers State University of New Jersey and UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. He is serving as an editorial board member of reputed journals and has reviewed manuscripts for more than twenty journals.

TGF-beta signaling Mammary stem cell function Cell cycle Tumor metastasis Breast cancer Prostate cancer

Laure Aurelian Professor Department of Pharmacology and Experimental Therapeutics The University of Maryland School of Medicine USA

1958-1962: Tel-Aviv University, Tel-Aviv, Israel. Awarded Master of Science Degree, June 1962. rn1962-1966: Graduate Work for the degree of Doctor of Philosophy. Department of Microbiology, The Johns Hopkins School of Medicine. rn1966: Degree of Doctor of Philosophy.

Oncology, Immunology and Genetic Vaccines.

Rita C. R. Perlingeiro Associate Professor Lillehei Heart Institute Department of Medicine University of Minnesota USA

Dr. Rita C. R. Perlingeiro received her Ph.D. at the University of Campinas (UNICAMP) in Campinas, Sao Paulo, Brazil. She completed her postdoctoral training in Stem Cell Biology at the Whitehead Institute, MIT, in Cambridge, MA. She started her own laboratory in the Department of Developmental Biology at the University of Texas Southwestern Medical Center in 2003. Currently, she is as an Associate Professor in the Department of Medicine, Cardiovascular Division, and a member of the Lillehei Heart Institute at the University of Minnesota, Twin Cities. She has authored over 30 research articles as well as a chapter, Regulation of Angiogenesis in Coronary Heart Disease: Clinical Pathological, Imaging and Molecular Profiles, to be in press by the end of this year. In 2008, Dr. Perlingeiro and colleagues published a seminal article, Functional skeletal muscle regeneration from differentiating embryonic stem cells (Nat. Med. 2008, 14:134-143). This was the first example of using embryonic stem cells to improve muscle function in muscular dystrophy. Such findings have extraordinary biological and therapeutic significance.

The main focus of the Perlingeiro laboratory is to understand the molecular mechanisms controlling lineage decision from early mesoderm towards skeletal muscle, blood, and endothelial cells, with the ultimate goal to generate specific cell types from ES and iPS cells for therapeutic applications.

Qing Ma Associate Professor of Cancer Medicine Department of Stem Cell Transplantation and Cellular Therapy University of Texas M.D. Anderson Cancer Center Houston, USA

Prof/Dr Qing Ma has received his PhD in Thomas Jefferson University during the period of 1990-1995. Currently, she is working as an Associate Professor of Cancer Medicine in the University of Texas M.D. Anderson Cancer Center. She has successfully completed his Administrative responsibilities as she is serving as an reviewer or editorial member of several reputed journals like Blood, Journal of Immunology, Biology of Blood and Marrow Transplantation, Journal of Biological Chemistry, Cancer Research, Journal of Leukocyte Biology, World Journal of Biological Chemistry , International Journal of Immunology Research. She has authored 23 research articles/books. She is a member of The American Association of Immunologists, The American Society of Hematology, American Society for Blood and Marrow Transplantation, The Society for Leukocyte Biology.She has honored as a Irvington Fellow and American Cancer Society Research Scholar.

Integrin, Chemokine, Stem cell transplantation, GVHD, GVL, Immunotherapy.

Min Du Associate Professor Department of Animal Science Developmental Biology Group, College of Agriculture University of Wyoming Laramie, USA

Min Du is the Leader of Development Biology Group, Department of Animal Science, Associate Professor in Muscle Biology, Associate Professor of Biomedical Program, Associate Professor of Molecular and Cellular Life Sciences, University of Wyoming. He has received a PhD in Muscle Biology from Iowa State University, Ames, IA in, 1998-2001. He has completed his M.S. in Muscle Biology in China Agricultural University, Beijing, China (1993). He has obtained his B.S. in Food Engineering in Zhejiang Agricultural University, Hangzhou, China (1990). He received Early Career Achievement Award, form American Society of Animal Science. He is serving as an associate editor for Journal of Animal Science, reviewer for more than 20 journals and several federal funding agencies. He has published more than 100 peer-reviewed papers in muscle biology.

Skeletal muscle development Mesenchymal stem cell differentiation Myogenesis Adipogenesis Fibrogenesis Cell signaling and gene expression Epigenetic modifications.

Elena Jones Associate Professor Academic Unit of Musculoskeletal Disease Leeds Institute of Molecular Medicine United Kingdom

Doctor Elena Jones is an Associate Professor in the Leeds Institute of Rheumatic and Musculoskeletal Medicine (LIRMM), the University of Leeds. She graduated with a BSc in Immunology and obtained a PhD in Experimental Oncology from the All-Union Cancer Research Centre, Russian Academy of Medical Sciences, Moscow. In Moscow she has developed Russia-first antibodies to human hematopoietic stem cells and B cells applicable for leukaemia diagnosis. In 1993 she obtained a prestigious Royal Society Postdoctoral Research Fellowship and arrived in HMDS, Leeds, where she considerably advanced her experience in bone marrowphenotyping using flow cytometry. She subsequently obtained a post-doctoral research position in the Molecular Medicine Unit, where she gained first experience with marrow stromal cells/MSCs. Her post-doctoral studies were dedicated to gene therapy with MSCs. Since joining the Academic Unit of Musculoskeletal Disease, her research interests are focused on the study of human MSCs in health and disease and their use in Regenerative Medicine. In 2002 she described the phenotype of native/uncultured MSCs in bone marrow and in 2004 she discovered MSCs in synovial fluid. Her MSC isolation methodology based on the CD271 marker has been adopted by the Industry, initially as research methodology and subsequently as a clinical-grade process. She has subsequently developed novel ideas on large-scale extraction of MSCs from bone, soft tissues (synovium and joint fat) and from surgical by-products (reaming waste bags and fatty marrow). She is currently working towards the therapeutic use of minimally-manipulated uncultured MSCs in bone repair applications including novel scaffolds and quality-control assays for cell manufacture. In relation to cartilage tissue regeneration her major interest lies in the use of endogenous synovial MSCs in combination with biomimetic scaffolds in patients with early osteoarthritis. She continues to explore the biology of synovial fluid MSCs including their homeostatic trafficking and therapeutic targeting to injured areas.

She is currently working towards the therapeutic use of minimally-manipulated uncultured MSCs in bone repair applications including novel scaffolds and quality-control assays for cell manufacture. In relation to cartilage tissue regeneration her major interest lies in the use of endogenous synovial MSCs in combination with biomimetic scaffolds in patients with early osteoarthritis. She continues to explore the biology of synovial fluid MSCs including their homeostatic trafficking and therapeutic targeting to injured areas.

Thomas Lufkin Associate Professor Stem Cell and Developmental Biology National University of Singapore Singapore 138672 Tel. 65 6808 8167 Fax 65 6808 8307

Thomas Lufkin is a Senior Group Leader in Stem Cell & Developmental Biology, Genome Institute of Singapore. He is Associate Professor, Department of Biological Science, National University of Singapore, Associate Professor for the School of Biological Science, Nanyang Technological University. He completed postdoctoral training at the LGME, Strasbourg, France, in Molecular Embryology (with Pierre Chambon). He received his Ph.D. from Cornell University in Molecular Biology and Virology. He received his A.B. from the University of California, Berkeley in Cell Biology. He received the March of Dimes Basil OConner Jr. Faculty Award, was a Lucille B. Markey Scholar in Molecular Biology, received an Alfred P. Sloan Research Fellowship in Neuroscience, an American Cancer Society Postdoctoral Fellowship and a Morton J. Levy Predoctoral Fellowship. He is serving as an editorial board member of 3 reputed journals. He has 74 publications.

Embryonic Stem Cell Differentiation Embryogenesis Developmental Genomics Gene regulatory networks Systems Biology Regenerative Medicine Vertebrate Development.

Rosalinda Madonna, MD, PhD Assistant Professor Internal Medicine, Cardiology Division University of Texas Medical School Houston, USA

Rosalinda Madonna is Assistant Professor, Internal Medicine, Cardiology Division, University of Texas Medical School (UT) in Houston and Research Scientist, Texas Heart Institute (THI) in Houston. She received her MD in University of Chieti, Italy (1997) and PhD in Biotechnology at the same University (2003). She completed her post-doctoral research fellowship in Molecular Cardiology (2007, University of Louisville, KY) and Atherosclerosis and Heart Failure (2002 2006, UT and THI Houston). She completed her Residency and Clinical Fellowship in Cardiology in University of Chieti (2003-2007). She has a Master in Internal Echocardiography and Cardio-Respiratory Physiopathology and stress test (in Centro Cardiologico Monzino, Milan, Italy) She is recipient of several awards and research grants (2003: Award for best abstract by The International Society of Thrombosis and Haemostasis; 2003: Young Investigator award by The Italian Society of Thrombosis and Haemostasis; 2004: Travel grant by Alliance of Cardiovascular Researchers and The Brown Institute; 2004: Travel grant by The European Association Study of Diabetes (EASD); 2006: Scholarship by Italian Society of Cardiology (SIC); 2007, 2008 and 2009 Scholarship by The National Institute for Cardiovascular Research; 2008 Scholarship by SIC and Sanofi Aventis; 2010 Travel grant young scientist by European Society of Cardiology (ESC). Ongoing reviewer of Circulation Research, Expert Reviews, Cardiovascular Research, Atherosclerosis, Journal of Molecular and Cellular Cardiology, Thrombosis and Haemostasis, Journal of Internal and Emergency Medicine, International Journal of Cardiology, The Journal of Diabetes Complications. Member of several International Societies and Nucleus Member ESC Working Group on Cellular Biology of the Heart. Author and co-author of 42 journal papers, 7 book chapters, 100 abstracts.

Stem cells, iPS cells, Cardiac development, Gene cloning and gene therapy, Biomaterials, Physiopathology of atherosclerosis in diabetes.

Morayma Reyes Assistant Professor Department of Pathology Department of Laboratory Medicine University of Washington Seattle, USA

She is an Assistant Professor for the Department of Pathology and Laboratory Medicine, Member of Institute for Stem Cell and Regenerative Medicine, Member of Center for Cardiovascular Biology, University of Washington. She has received her MD/PhD degree from University of Minnesota, 1996-2003. She has completed her B.S. in biology and chemistry from the University of Puerto Rico, 1996. She is serving as an editorial board member of reputed journals and reviewer of 3 journals. She has been nominated and awarded for the Princeton Global Networks and the Madison Whos Who Member-Executives and Professionals. She received the Junior Faculty Awards: Perkins Coie Award and the Marian E. Smith award.

Adult stem cells Skeletal muscle and heart regeneration Stem cell therapy for muscular dystrophy Stem cell homing and migration Tissue regeneration/ Bioengineering/ artificial organs Mesenchymal stem cells Dental Pulp Stem Cells Vascular Biology Hemostasis/ thrombosis/ Coagulation Angiogenesis.

Ipsita Banerjee Assistant Professor Department of Chemical Engineering McGowan Institute for Regenerative Medicine University of Pittsburgh Pittsburgh, USA

Ipsita Banerjee is a faculty in Chemical Engineering department of University of Pittsburgh. Adjunct faculty of Bioengineering Department, University of Pittsburgh. Adjunct faculty of McGowan Institute for Regenerative Medicine. She has completed three years of post-doctoral training in Harvard Medical School, Boston, MA, (2005-2008). She received her PhD from Rutgers University, NJ (2000-2005). She received the NIH New Innovator Award and the Ralph Powe Faculty Enhancement Award. She currently has fourteen publications in reputed international journals. She is a reviewer for Tissue Engineering, Tissue Engineering and Regenerative Medicine, Journal of Biotechnology, Computers and Chemical Engineering, Journal of Integrative Biology, Cellular and Molecular Bioengineering. She serves on the review panel of National Science Foundation, Biomedical Engineering Division.

Embryonic stem cell differentiation iPS cell differentiation Diabetes Systems Biology Analysis of regulatory network of differentiating stem cells Optimization based algorithm for network identification Agent Based Modeling for differentiation patterning.

Porrata Luis F Assistant Professor and Assistant Deputy Director of the Blood and Marrow Program Mayo Clinic Transplant Center Rochester, USA

Luis F. Porrata is Assistant Deputy Director of the Blood and Marrow Program, Mayo Clinic. Assistant Professor, Division of Hematology, Department of Medicine, Mayo Clinic. He is serving as an editorial board member of reputed journals and reviewer of several journals including Blood, Bone Marrow Transplantation, and Biology of Blood and Marrow transplantation.

Autologous stem cell transplantation Lymphoma Immunotherapy.

Yoon-Young Jang Assistant Professor Stem Cell Biology Laboratory Johns Hopkins Medical Institutions Baltimore, USA

Yoon-Young Jang, MD, PhD is a Assistant Professor of Stem Cell Biology Laboratory, Oncology at Johns Hopkins University School of Medicine, Baltimore, Maryland. She has received MD, PhD from the Chung-Ang University, Seoul, Korea and has completed fellowpship in Johns Hopkins University. She been a faculty member at Johns Hopkins Oncology Center since 2005 and has awarded three stem cell grants from the Maryland State.

Stem cell biology (Pluripotent stem cells, Cancer stem cells, Hematopoietic stem cells) Hepatic differentiation of human stem cells Liver regeneration using animal models of liver diseases Disease modelling using iPS derived hepatocytes Stem cell niche biology

Yong Zhao Assistant Professor Section of Diabetes and Metabolism Department of Medicine University of Illinois Chicago, USA

Yong Zhao, MD, PhD, Assistant Professor, Section of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Illinois at Chicago. He received his PhD (2000) in Immunology at Shanghai Second Military Medical University, Shanghai, China. He received his MD (1990) in Clinical Medicine at Weifang Medical College, Shandong, China. He received 2006 and 2008 Rachmiel Levine Scientific Achievement Award. He has 24 peer-reviewed publications. He owned 8 patents.

Umbilical cord blood stem cells Hematopoietic stem cells Immune modulation Type 1 diabetes Type 2 diabetes Pancreatic islet beta cell differentiation Humanized mice

Chia-Ying Lin Research Assistant Professor Director, Spine Research Laboratory University of Michigan Ann Arbor, USA

Chia-Ying Lin is a Research Assistant Professor, the Director of the Spine Research Laboratory at the Department of Neurosurgery in the University of Michigan. He has received his MS and PhD in Biomedical Engineering from the University of Michigan, Ann Arbor, MI, in 2002 and 2004, respectively. He has completed his B.A in Civil Engineering in National Taiwan University, Taipei, Taiwan in 1997. Dr. Lin is serving as an editorial board member of reputed journals and reviewer of 6 journals, including Biomacromolecules, Tissue Engineering, Journal of Biomedical Materials Research, Materials Letters, Cell Proliferation, and Journal of Orthopaedic Research. He has published over 20 articles to date in many journals specified in spine medicine, regenerative medicine, and cancer biology and therapy.

His research interests primarily focus on biological repair of degenerative intervertebral disc, spinal reconstruction with tissue engineering approaches, and inductive therapy for bone metastasis.

Tonya J. Roberts Webb Assistant Professor Department of Microbiology and Immunology Member of the Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine, USA

Tonya J. Roberts Webb completed Ph. D in 2003 and serving as Assistant Professor in Department of Microbiology and Immunology, University of Maryland School of Medicine.

Microbiology and Immunology.

Vincenzo Lionetti Assistant Professor of Physiology Sector of Medicine Scuola Superiore Sant Anna University Pisa, Italy Tel. 39-328-0078806 Read Interview session with Vincenzo Lionetti

Vincenzo Lionetti is Head of Unit of Molecular and Translational Medicine, National Institute of Biostructures and Biosystems, Bologna, Italy; Assistant Professor of Physiology, Sector of Medicine, Scuola Superiore SantAnna, Pisa, Itay; Adjunct Researcher, Institute of Clinical Physiology, National Council of Research, Pisa, Italy. Adjunct Researcher, Fondazione Regione Toscana Gabriele Monasterio, Pisa, Italy. He has received a PhD in Innovative Strategies in Biomedical Research from the Scuola Superiore SantAnna, Pisa, Italy, in 2007. He has specialized in Anesthesiology and Intensive Care Medicine at the University of Turin, Italy, in 2003. He received: Trainee Abstract Award from the Council on Basic Cardiovascular Sciences of the American Heart Association in 2002; Young Investigator Award from the National Institute of Cardiovascular Research in 2009. He is serving as a member of the Council on Cardiovascular Science of the American Heart Association and Study Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology. He is serving as peer reviewer for Cardiovascular Research, Ultrasound in Medicine and Biology, ECAM, Clinical Journal of the American Society of Nephrology, American Journal of Physiology-Heart and Circulatory Physiology. He has published 5 book chapters; 22 peer-reviewed articles in international journals including: Journal of Biological Chemistry, Cardiovascular Research, Journal of Cardiac Failure, American Journal of Physiology, Journal of Physiology (London), Journal of Molecular and Cellular Cardiology, FASEB Journal.

Physiology and physiopathology of regenerate myocardium Regional imaging of regenerate myocardium Physiopathology of heart failure Innovative acellular therapies to repair failing myocardium.

Rajasingh Johnson Assistant Professor Department of Medicine Cardiovascular Research Institute University of Kansas Medical Center, Kansas City, USA

Dr.Rajasingh Johnson has received his PhD in Vanderbilt University during the period of 2004-2007. Currently, he is working as Assistant Professor in University of Kansas Medical Center.

My research interests include the de-differentiation of somatic cells by chromatin modifying agents to generate induced pluripotent (iPS cells) or multipotent stem cells and its therapeutic potential in regenerative medicines; mechanisms of somatic cell reprogramming by histone deacetylation and DNA methylation inhibitors; differentiation of embryonic and adult stem cells in cardiovascular and lung vascular repair and regeneration.

Prasanna Krishnamurthy, DVM, PhD Assistant Professor Feinberg School of Medicine Cardiovascular Research Institute Northwestern University, Chicago, USA

Dr. Prasanna (Krish) Krishnamurthy received his PhD in Indian Veterinary Research Institute during the period of 2000-2003. Currently, he is working as Assistant Professor in Northwestern University.

My research interests include endothelial progenitor cell, myocardial ischemia, cell-based regenerative therapy for heart failure and bone marrow transplantation.

Atsushi Asakura Assistant Professor Department of Neurology University of Minnesota Medical School MN 55455, USA

Li Xiao Assistant Professor Department of Pharmacology The Nippon Dental University, Tokyo, Japan

Dr. Li Xiao has received her PhD in Prefectural University of Hiroshima in the year 2007. Currently, she is working as Asssistant Professor in The Nippon Dental University.

Research interests includes tissue engineering, antioxidant, radiation Biology, regenerative medicine and traditional Chinese medicine.

Raji Padmanabhan Research Scientist Laboratory of Cell Biology (LCB) Center for Cancer Research (CCR) National Cancer Institute(NCI) National Institutes of Health, (NIH)Bethesda Maryland 20892,USA Tel. (301) 496-3096 Read Interview session with Raji Padmanabhan

Richard Schaefer Department of Stem Cell and Regenerative Biology Harvard Stem Cell Institute Harvard University Cambridge, USA

Dr. Richard Schaefer, MD is the head of the Mesenchymal Stem Cell Laboratory, Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tuebingen, Germany. Research Fellow at the Department of Stem Cell and Regenerative Biology Harvard University, Cambridge, USA. Specialist for Internal Medicine and Transfusion Medicine. After studying Medicine in Giessen, Germany and Mannheim/Heidelberg, Germany he has received his MD in 1997. He is serving as an editorial board member of reputed journals and reviewer of 12 journals. He is author of more than 20 articles published in international journals and co-editor of the Handbook of Stem Cell Based Tissue Repair Royal Society of Chemistry, Cambridge, U.K.

Stem Cell Biology Characterization, Differentiation, Immunomodulation Mesenchymal (Stem/Stromal) Cells Regenerative Medicine Labeling and Imaging of Stem Cells GMP production of cellular therapies.

Christian Drapeau, PhD StemTech HealthSciences, LLC 1011 Calle Amanecer San Clemente, California, USA

1991 Master degree in Neurology and Neurosurgery from McGill University, Montreal,Quebec, Canada. Work performed at the Montreal Neurological Institute.Thesis on epileptogenesis and the role of eicosanoids in long-term potentiation.1987 Bachelor degree in Honors Neurophysiology from McGill University, Montreal, Quebec, Canada. Program limited to 6 students.

Neurology and Neurophysiology.

Shi-Jiang Lu, PhD, MPH Senior Director for Research Advanced Cell Technology Marlborough, USA Read Interview session with Shi-Jiang Lu

Shi-Jiang Lu is currently a Senior Director of Stem Cell and Regenerative Medicine International, a joint venture between Advanced Cell Technology and CHA Biotech of Korea; Adjunct Professor, Department of Applied Bioscience, Cha University, Seoul, Korea, and Scientific Advisor, Advanced Cell Technology, Inc., Marlborough, MA. He was Senior Director, Director and Senior Scientist, Advanced Cell Technology, Inc., Marlborough, MA, and Director and Assistant Professor, Stem Cell Research Program, Department of Pediatrics, University of Illinois at Chicago, Chicago, IL. He received a PhD in Molecular Biology and Cancer from Department of Medical Biophysics, University of Toronto, Toronto, Canada (1992). He completed his MPH from School of Public Health, Columbia University in New York (1988) and MSc from Peking Union Medical College, Beijing, China (1985). He received a BS in Biochemistry from Wuhan University, Hubei, China (1982). He has more than 50 publications and Book Chapters.

Stem Cells: embryonic stem cells (ES), induced pluripotnet stem cells (iPS), and hematopoietic stem cells (HSC), cancer stem cells, ES and iPS cell lineage specific diffeentiation. Hematopoietic Cells: bone marrow transplantation, red blood cells, megakaryocytes and platelets.Stem Cell therapy: ischemic vessel lesions and stem cell treatment, diabetic retinopathy and stem cell treatment, cardiomyocyte infarction and stem cell treatment.

Alex F. Chen, MD, PhD, FAHA Director Department of Surgery University of Pittsburgh School of Medicine Pittsburgh, USA

Alex F. Chen is Director of VA Vascular Surgery Research, and an Associate Professor, Department of Surgery, University of Pittsburgh School of Medicine. He has received a MD from Hunan Medical University in 1985 and a PhD in Pharmacology from Southern Illinois University in 1995. He is serving as an editorial board member of several reputed journals.

Vascular and endothelial cell biology Endothelial progenitor cells Redox regulation of endothelial function in diabetes and hypertension.

Alastair Wilkins Senior Lecturer Neurology Consultant Neurologist University of Bristol Bristol, UK

Alastair Wilkins is Senior Lecturer in Neurology, University of Bristol and Head of Neurology, Frenchay Hospital, Bristol, UK. He received a PhD in Clinical Neuroscience from the University of Cambridge in 2003. He has completed his B.A in Medical Sciences and MB BChir from the University of Cambridge in 1993. He is a fellow of the Royal College of Physicians (UK). He has published more than 40 articles, including reviews and book chapters. His Current research projects includes role of the peroxisome in axonal degeneration and progressive MS, developing a model of secondary progressive MS (taiep rat), degenerative ataxias and the potential for stem cell neuroprotection, developing Growth factor therapies for progressive multiple sclerosis, analysis of VLCFAs in serum of patients with multiple sclerosis, analysis of Reactive Oxygen Species in Multiple Sclerosis cerebrospinal fluid, local investigator for the analysis of genetic factors in multiple sclerosis (PI: Prof Alastair Compston, University of Cambridge)

Multiple sclerosis Neurobiology of axon degeneration Applications of neuroreparative stem cell therapies.

James Adjaye Department of Vertebrate Genomics Molecular Embryology and Aging Group Max Planck Institute for Molecular Genetics Ihnestrasse 73, D-14195 Berlin, Germany

James Adjaye is a Group Leader at the Max-Planck Institute for Molecular Genetics (Molecular Embryology and Aging group).Adjunct Associate Professor for stem cell biology, College of Medicine Stem Cell Unit, King Saud University, Riyad, Saudi Arabia. He has received a PhD in biochemistry at Kings College London 1992. He has completed his BSc studies in biochemistry at University College Cardiff, Wales 1987. He is serving as an editorial board member of 4 reputed journals and reviewer of 17 journals.

Transcriptional and signal transduction mechanisms regulating self renewal and pluripotency in human embryonic stem cells, embryonal carcinoma cells and iPS cells (induced pluripotent stem cells). Reprogramming of somatic cells (healthy and diseased individuals- Alzheimers, Diabetic, Nijmegen breakage syndrome and Steatosis patients) into an ES-like state (iPS cells) and studying the underlying disease mechanisms. Systems biology of stem cell fate and cellular reprogramming.

Stefano Biressi Post-doctoral research associate Department of Neurology and Neurological Sciences Stanford University USA

He studied at the University of Milan, Italy. He received his PhD in Cellular and Molecular Biology from The Open University of London. He worked in the Telethon Institute for Gene Therapy (TIGeT) and in the Stem Cell Research Institute, Hospital San Raffaele, Milan, Italy. He is currently working in the Department of Neurology and Neurological Sciences at Stanford University, CA, USA.

Cellular and molecular mechanisms regulating skeletal muscle development, regeneration and muscle stem cells self-renewal and lineage progression in normal and pathological conditions.

Hosam A. Elbaz Department of Basic Pharmaceutical Sciences West Virginia University Morgantown, USA Read Interview session with Hosam A Elbaz

Dr Hosam A. Elbaz has received his PhD in West Virginia University during the period of 2007 2011. Currently, he is working as a postdoctoral fellow in Wayne State University School of Medicine. He is serving as an editorial member for several reputable journals like Journal of Bioengineering and Biomedical Sciences, Journal of Nanomedicine and Nanotechnology, Pharmaceutica Analytica Acta, and Biochemistry and Pharmacology. He is a member of American Society of Pharmacology and Experimental Therapeutics (ASPET), American Association of Pharmaceutical Scientists (AAPS), American Chemical Society (ACS), Egyptian General Syndicate of Pharmacists, and Golden Key International Honor Society.

Cancer Therapeutics,Carcinogenesis, Cell Cycle and Checkpoint Regulation, Apoptosis, Nanomedicine and Nanobiotechnology, Targeted Drug Delivery, Therapeutic Gene Delivery, Biochemical Pharmacology and Toxicology.

Amir Hamdi, MD Postdoctoral research fellow Department of Stem Cell Transplantation and Cellular Therapy The University of Texas MD Anderson Cancer Center Houston, Texas, USA

Dr. Amir Hamdi was born and raised in Iran. He received his M.D. degree from Tabriz University of Medical Sciences. He was a research scientist in Hematology, Oncology and Stem Cell Transplantation Research Center in Tehran and participated in several research projects. He is currently a postdoctoral research fellow in the Department of Stem Cell Transplantation and Cellular Therapy at The University of Texas MD Anderson Cancer Center.

Dr. Hamdis research interests include therapy of leukemias and lymphomas as well as development of investigational approach for the treatment of hematologic and neurologic disorders. He has published several papers related to neurology, hematology, oncology and stem cell transplantation; and serves as reviewer for various journals.

Haigang Gu Postdoctoral Fellow Vanderbilt University School of Medicine Nashville, USA Read Interview session with Haigang Gu

Haigang Gu, cuurently Postdoctoral researcher in Vanderbilt University School of Medicine, Nashville, USA. Haigang Gu has received his PhD in also in Emory University during the period of 2010-2011.

My current research is to understand how transcriptional factors affect neuronal differentiation and maturation and synaptic transmission and recycling in vitro and in vivo using stem cell-derived neurons, primary cultured neurons and brain slices by whole cell patch clamp recording and super-resolution live cell imaging. The underlying mechanisms could be extended to illustrate the functional recovery of neurological disease treated by drugs and stem cells. Recently, I have cloned most of neuronal transcriptional factors (15 genes) in lentiviral-based vector and packaged these vectors in lentivirus. We have developed some new protocols to induce stem cells, embryonic stem cells and neural stem cells to differentiate into neurons using defined chemicals and transcriptional factors related to neuronal differentiation and maintenance. Furthermore, we have made substantial progress on the synaptic transmission and recycling trafficking in cultured hippocampus, cortical and midbrain neurons. My research has been mainly focus on understanding (1) the mechanisms of proliferation and neuronal differentiation of embryonic stem cells and adult stem cells, such as neural stem cells and mesenchymal stem cells, (2) stem cell-based therapies for the treatment of such as Alzheimers disease and ischemic stroke, and (3) sustained release neurotrophic factors or neurotrophic factor genes for the treatment of neurodegenerative disease. I have strong background and extensive experience in molecular and cellular biology, stem cell culture and differentiation, whole cell patch clamp recording in cultured cells, live cell imaging as well as animal models, such as Parkinsons, Alzheimers disease and ischemic stroke.

Dhanajaya Nayak Department of Biochemistry University of Wisconsin-Madison USA

Dr. Dhanajaya Nayak (PhD) currently holds an Assistant Scientist position in the Department of Biochemistry at University of Wisconsin-Madison (2013-present). Previously, he has received a master of technology (M.Tech.) degree from the Indian Institute of Technology, Kharagpur, India, and a PhD degree in Biochemistry from the University of Texas Health Science Center at San Antonio (2004-2009), where he won the prestigious Armand J. Guarino Award for academic excellence in doctoral studies in Biochemistry. After his PhD, he joined the Department of Biochemistry at University of Wisconsin-Madison as a postdoctoral research associate (2009-2012). Dr. Nayak has more than 8 years of research experience in the field of transcription and gene regulation. He is a member of the American Association for the Advancement of Science (AAAS) and International Society for Cardiovascular Translational Research (ISCTR). At present, he is an active reviewer for several journals from the OMICS group: Journal of Stem Cell Research and Therapy, Journal of Enzyme Engineering, Journal of Molecular Biomarkers and Diagnosis, Journal of Chemical Engineering and Process Technology and Journal of Analytical and Bioanalytical Technique etc

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Stem Cells Therapy IPS Cell Therapy IPS Cell Therapy

[Retinal Cell Therapy Using iPS Cells].

Progress in basic research, starting with the work on neural stem cells in the middle 1990’s to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author’s group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient’s own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment is now being investigated in preclinical research. Among the mixture of rod and cone photoreceptors in the donor cells, the percentage of cone photoreceptors is still low. Donor cells with more. cone photoreceptors will be needed. If that will work well, photoreceptor transplantation will be the first example of neural network reconstruction in the central nervous system. These efforts will reach to variety of retinal cell transplantations in the future.

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[Retinal Cell Therapy Using iPS Cells].

stem cells – The ALS Association

Quick links:

Stem cells are cells that have the ability to divide for indefinite periods in culture and give rise to multiple specialized cell types. They can develop into blood, bone, brain, muscle, skin and other organs.

Stem cells occur naturally, or they can be created from other kinds of cells. Stem cells form during development (embryonic stem cells). They are also present in small numbers in many different tissues (endogenous adult stem cells). Most significantly, stem cells can be created from skin cells (induced pluripotent stem cells, or iPS cells).

iPS cells have emerged in recent years as by far the most significant source of stem cells for ALS research. A simple skin biopsy provides the skin cells (fibroblasts). These cells are treated in a lab dish with a precise cocktail of naturally occurring growth factors that turns back the clock, transforming them back into cells much like those that gave rise to themstem cells.

Embryonic stem cells can be isolated from fertilized embryos less than a week old. Before the development of iPS cells, human embryos were the only source of human stem cells for research or therapeutic development. The ethical issues involved hindered development of this research. Most stem cell research in ALS is currently focused on iPS cells, which are not burdened with these issues.

Stem cells are being used in many laboratories today for research into the causes of and treatments for ALS. Most commonly, iPS cells are converted into motor neurons, the cells affected in ALS. These motor neurons can be grown in a dish and studied to determine how the disease develops. They can also be used to screen for drugs that can alter the disease process. The availability of large numbers of identical neurons, made possible by iPS cells, has dramatically expanded the ability to search for new treatments.

Because iPS cells can be made from skin samples of any person, researchers have begun to make individual cell lines derived from dozens of individuals with ALS. Comparing the motor neurons derived from these cells lines allows them to ask what is common, and what is unique, about each case of ALS, leading to further understanding of the disease process.

Stem cells may also have a role to play in treating the disease. The most likely application may be to use stem cells or cells derived from them to deliver growth factors or protective molecules to motor neurons in the spinal cord. Clinical trials of such stem cell transplants are in the early stages, but appear to be safe.

While the idea of replacing dying motor neurons with new ones derived from stem cells is appealing, there are multiple major hurdles that must be overcome before it is a possibility. Perhaps the most challenging is coaxing the implanted cells to grow the long distances from the spinal cord, where they would be implanted, out to the muscle, where they cause contraction. While work is ongoing to overcome these challenges, it is likely that providing support and protection to surviving neurons represents a more immediate possible form of stem cell therapy.

The presence of endogenous stem cells in the adult brain and spinal cord may provide an alternative to transplantation, eliminating the issues of tissue rejection. If there were a way to stimulate resident stem cells to replace dying cells the limitations of transplantation could be overcome. Small biotech companies are pursuing this direction in the hope of finding therapeutic compounds that will do this. Further research into molecules and genes that govern cell division, migration and specialization is needed, ultimately leading to new drug targets and therapies for ALS.

The mechanism of motor neuron death in ALS remains unclear. It is not certain that transplanted stem cells would be resistant to the same source(s) of damage that causes motor neurons to die and stem cells may need to be modified to protect against the toxic environment. There is also the potential that cultured stem cells used in transplant medicine could face rejection by the body’s immune system.

The NeuralStem trial demonstrated the safety of transplanting human embryonic stem cells into the spinal cord of people with ALS. As of late 2014, a larger trial of the same technique is underway, to determine whether treatment can improve function or slow decline. More information can be found here: http://www.alsconsortium.org/news_neuralstem_phaseII_first_patient.php

The BrainStorm trial is underway as of late 2014, examining the safety and efficacy of transplantation of autologous mesenchymal stem cells secreting neurotrophic factors. These stem cells are extracted from the patients own bone marrow, then treated to increase their production of protective factors, and then injected into muscle and the region surrounding the spinal cord. More information can be found here: http://www.alsconsortium.org/

Read The ALS Associations Statement on Stem Cell Research.

Last update: 08/26/14

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stem cells – The ALS Association

Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for …

Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3′-untranslated region (3′ UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.Molecular Therapy (2016); doi:10.1038/mt.2016.97.

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Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for …

What are iPS cells? | For the Public | CiRA | Center for …

Research is ongoing in Japan and overseas with the aim of realizing cell transplantation therapy using iPS cells. One safety issue of concern is the risk of tumor formation. CiRA in particular has focused its resources on this issue.

Broadly speaking, there are two main theories as to the mechanism whereby iPS cells may form tumors. One theory is that iPS cells form tumors in response either to reactivation of the reprogramming factors inserted into the cell or through damage caused to the original cell genome through the artificial insertion of the reprogramming factors. In response, a search was launched for optimal reprogramming factors which do not cause reactivation, and a method of generating iPS cells was developed in which reprogramming factors are not incorporated into the cell chromosomes and damage to the host genome is therefore avoided.

The other theory is that residues of undifferentiated cells – cells which have not successfully completed differentiation to the target cell type – or other factors lead to the formation of teratomas, a kind of benign tumor. This theory requires research on iPS cell proliferation and differentiation.

1. Search for optimal reprogramming factors When Professor Shinya Yamanaka and his research team announced the successful generation of mouse iPS cells, one of the reprogramming factors they used was c-Myc, which is known to be an oncogene, that is a cancer-causing gene. There have been suggestions that this gene may be activated within the cell and cause a tumor to form. However, in 2010, CiRA Lecturer Masato Nakagawa and his team reported that L-Myc was a promising replacement factor for c-Myc. iPS cells created using L-Myc not only display almost no tumor formation, they also have a high rate of successful generation and a high degree of pluripotency.

2. Search for optimal vectors When the reprogramming factors required to generate iPS cells were inserted into the cells of the skin or other body tissues, early methods employed a retrovirus or lentivirus as a “vector,” or carrier. In these methods, the target genes are inserted into the viruses with the which the cells were then infected in order to deliver the target genes. When a retrovirus or lentivirus is used as a vector, however, the viruses are incorporated into the cells genomic DNA in a random fashion. This may cause some of the cells original genes to be lost, or in other cases activated, resulting in a risk of cancerous changes.

In 2008, to remedy this risk, CiRA Lecturer Keisuke Okita and his team explored the use of a circular DNA fragment known as a plasmid, which is not incorporated into the cell chromosome, as a substitute to the retrovirus or lentivirus methods. In this way, they developed a method of generating iPS cells in which the reprogramming factors are not incorporated into the cell chromosome. In 2011, Okita and his team further improved the efficiency generation by introducing into a self-replicating episomal plasmid six factors – OCT3/4, SOX2, KLF4, LIN28, L-MYC, and p53shRNA.

3. Establishing a method for generating and screening safe cells Once iPS cells have been induced to differentiate into the target somatic cells using the appropriate genes and gene insertion methods as explained above, the differentiated cells can be relied upon not to revert to the undifferentiated state. However, there may sometimes be a residue of undifferentiated cells which have not completed the process of differentiation into the target cells, and it is possible that these cells, however few, may form a tumor. Scientists had already established that different iPS cell lines, even if generated from the same individual using the same method, might nevertheless display differences in proliferation and differentiation potentials. This meant that, if iPS cells with low differentiation potential were used, there was a risk that a residue of cells in the cell group might fail to fully differentiate and result in the formation of a teratoma. In 2013, a team led by CiRA Lecturer Kazutoshi Takahashi and Dr. Michiyo Aoi, now an assistant professor at Kobe University, developed a simple method to screen for iPS cell lines that have high potential to differentiate into nerve cells. There is also a risk of tumorigenesis from genomic or other damage arising at the iPS cell generation stage or at the subsequent culture stage. CiRA Assistant Professor Akira Watanabe and his team have developed a sensitive method to detect genomic and other damage in iPS cells using the latest equipment.

4. Developing a reliable method of differentiation into the target cell type In cell transplantation therapy, iPS cells are not transplanted directly into the human body. Instead, cells are transplanted after first being differentiated into the target cell type. It is therefore important to develop a reliable method of inducing iPS cells to differentiate into the target cell type. CiRA is currently working to develop technology for differentiation into a range of different cell types from iPS cells. CiRA Professor Jun Takahashi and his team have developed a highly efficient method of inducing iPS cells to differentiate into dopamine-producing nerve cells. In 2014, CiRA Professor Koji Eto and his team reported a method of producing platelets from iPS cells that is both reliable and can yield high volumes. These findings represent a major step toward iPS cell-based regenerative medicine for nerve diseases such as Parkinsons disease and blood diseases such as aplastic anemia.

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What are iPS cells? | For the Public | CiRA | Center for …

Regenerative medicine IPS Cell Therapy IPS Cell Therapy

Our Associate Medical Director, Professor Jeremy Pearson,discusses the parallels between todays news about treating paralysis and our hopes for mending broken hearts.

21 October 2014

This morning I woke to the news that a paralysed man could walk again. A medical miracle had been performed thanks to laboratory and clinical research. But when you break down the scientific journey thats got us to this point, you realise it isnt a miracle at all but decades of dedication and excellent science. This is the journey our funded researchers are on now as they work towards repairing and regenerating hearts damaged by heart attack.

A University College London (UCL) researcher, Professor Geoffrey Raisman, has been the driving force behind the paralysis breakthrough. Back in 1985 he discovered special cells in the nose that have a unique ability for allowing new nerve cells to grow. Almost three decades later, after developing a technique through studies in rats, we now have a potential treatment to regenerate a severed spinal cord.

This breakthrough is an excellent example of how persistence pays off in medical research. Laboratory science youre helping us to fund now could become a patient treatment in the future but the researchers need time and they need continued funding.

Professor Raisman was searching for a solution to a problem that seemed unsolvable something that our funded researchers can relate to. Right now, once a heart is damaged, like the spinal cord, it cannot be repaired. The heart doesnt spontaneously repair itself. A damaged heart cant pump blood around the body as well as it should, which can lead to heart failure. Heart failure can be severely disabling and prevent people carrying out basic tasks like going to the shops or washing without becoming totally exhausted.

Right now a BHF Professor Paul Riley(pictured) is moving us closer to a solving our unsolvable problem. In 2011, while at UCL, Professor Riley showed in mice how heart muscle can be regenerated in the adult heart after damage. Now at Oxford, he and his team are further investigating the outer layer of the heart, where these regenerative heart cells lie. We hope this work will eventually lead to a treatment that could be given to people after a heart attack to trigger the repair of any damage and prevent heart failure.

Due to difficulties in securing funding Professor Raismans progress was perhaps delayed by many years. With your support we hope to accelerate the progress that Professor Riley and his fellow researchers are making. We have already committed to funding 7.5 million across three Centres of Regenerative Medicine, one led by Professor Riley, that bring top researchers together with a common aim of repairing damaged heart muscle and blood vessels. And now we hope to raise a further 10 million towards a dedicated regenerative medicine facility for Professor Riley and his colleagues at Oxford.

This facility, called the Institute of Developmental and Regenerative Medicine, will bring experts from three separate disciplines under one roof where they can share facilities, ideas and resources making new treatments a reality much sooner.

Continue reading here: Regenerative medicine

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DNA repair Wikipedia, the free encyclopedia IPS Cell …

DNA damage resulting in multiple broken chromosomes

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.[1] Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cells ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cells genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages (interstrand crosslinks or ICLs).[2][3]

The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:

The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA damage repair and protection.[4]

The 2015 Nobel Prize in Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar for their work on the molecular mechanisms of DNA repair processes.[5][6]

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.[1] While this constitutes only 0.000165% of the human genomes approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cells ability to carry out its function and appreciably increase the likelihood of tumor formation and contribute to tumour heterogeneity.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around packaging proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

DNA damage can be subdivided into two main types:

The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for example, through gene conversion).

There are several types of damage to DNA due to endogenous cellular processes:

Damage caused by exogenous agents comes in many forms. Some examples are:

UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift.

In human cells, and eukaryotic cells in general, DNA is found in two cellular locations inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells.

Senescence, an irreversible process in which the cell no longer divides, is a protective response to the shortening of the chromosome ends. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (see Hayflick limit).[10] In contrast, quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the organism,[11] which serves as a last resort mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis is considered to be part of a strategy of protection against cancer.[12]

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damages and mutation are fundamentally different. Damages are physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damages can be recognized by enzymes, and, thus, they can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein will also be blocked. Replication may also be blocked or the cell may die.

In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce. Although distinctly different from each other, DNA damages and mutations are related because DNA damages often cause errors of DNA synthesis during replication or repair; these errors are a major source of mutation.

Given these properties of DNA damage and mutation, it can be seen that DNA damages are a special problem in non-dividing or slowly dividing cells, where unrepaired damages will tend to accumulate over time. On the other hand, in rapidly dividing cells, unrepaired DNA damages that do not kill the cell by blocking replication will tend to cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are deleterious to a cells survival. Thus, in a population of cells composing a tissue with replicating cells, mutant cells will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism, because such mutant cells can give rise to cancer. Thus, DNA damages in frequently dividing cells, because they give rise to mutations, are a prominent cause of cancer. In contrast, DNA damages in infrequently dividing cells are likely a prominent cause of aging.[13]

Single-strand and double-strand DNA damage

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when non-essential genes are missing or damaged). Depending on the type of damage inflicted on the DNAs double helical structure, a variety of repair strategies have evolved to restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort.

Damage to DNA alters the spatial configuration of the helix, and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place.

Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can occur in only one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300500nm wavelength) to promote catalysis.[14] Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans,[15] who instead use nucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used only once; that is, the reaction is stoichiometric rather than catalytic.[16] A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes.[17] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.[16]

Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination.[16] PVN Acharya noted that double-strand breaks and a cross-linkage joining both strands at the same point is irreparable because neither strand can then serve as a template for repair. The cell will die in the next mitosis or in some rare instances, mutate.[2][3]

In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends.[21] To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate.[22][23][24][25] NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are backup NHEJ pathways in higher eukaryotes.[26] Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.[27]

MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions.[28] In further steps,[29] PARP1 is required and may be an early step in MMEJ. There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. This is followed by recruitment of XRCC1LIG3 to the site for ligating the DNA ends, leading to an intact DNA.

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ).[30] In an in vitro system, MMEJ occurred in mammalian cells at the levels of 1020% of HR when both HR and NHEJ mechanisms were also available.[28] MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.[31]

Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.

Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNAs state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.

A team of French researchers bombarded Deinococcus radiodurans to study the mechanism of double-strand break DNA repair in that bacterium. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.[32]

Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.[33] It involves switching out regular DNA polymerases for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol mediates error-free bypass of lesions induced by UV irradiation, whereas Pol introduces mutations at these sites. Pol is known to add the first adenine across the T^T photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or repairing lesions at locations of stalled DNA replication. For example, Human DNA polymerase eta can bypass complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although can cause targeted and semi-targeted mutations.[34] Paromita Raychaudhury and Ashis Basu[35] studied the toxicity and mutagenesis of the same lesion in Escherichia coli by replicating a G[8,5-Me]T-modified plasmid in E. coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication.[36][37] After translesion synthesis, extension is required. This extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol , yet if TLS results in a mismatch, a specialized polymerase is needed to extend it; Pol . Pol is unique in that it can extend terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol to fix the lesion, then PCNA may switch to Pol to extend the mismatch, and last PCNA will switch to the processive polymerase to continue replication.

Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage.[38] The global response to damage is an act directed toward the cells own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.

After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,[39] whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified.[40] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

DNA damage checkpoint is a signal transduction pathway that blocks cell cycle progression in G1, G2 and metaphase and slows down the rate of S phase progression when DNA is damaged. It leads to a pause in cell cycle allowing the cell time to repair the damage before continuing to divide.

Checkpoint Proteins can be separated into four groups: phosphatidylinositol 3-kinase (PI3K)-like protein kinase, proliferating cell nuclear antigen (PCNA)-like group, two serine/threonine(S/T) kinases and their adaptors. Central to all DNA damage induced checkpoints responses is a pair of large protein kinases belonging to the first group of PI3K-like protein kinases-the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia- and Rad-related) kinases, whose sequence and functions have been well conserved in evolution. All DNA damage response requires either ATM or ATR because they have the ability to bind to the chromosomes at the site of DNA damage, together with accessory proteins that are platforms on which DNA damage response components and DNA repair complexes can be assembled.

An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage.[41] The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent kinase complexes.[42]

The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes.[43] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes.[44] The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed by DNA helicase to separate the two DNA strands.[38] In the initiation step, RecA protein binds to ssDNA in an ATP hydrolysis driven reaction creating RecAssDNA filaments. RecAssDNA filaments activate LexA autoprotease activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division and an increase in levels of proteins responsible for damage processing.

In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably, with different length and composition, but it is always highly conserved and one of the strongest short signals in the genome.[44] The high information content of SOS boxes permits differential binding of LexA to different promoters and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response. The error-prone translesion polymerases, for example, UmuCD2 (also called DNA polymerase V), are induced later on as a last resort.[45] Once the DNA damage is repaired or bypassed using polymerases or through recombination, the amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene expression.

Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage differently indicating an absence of a common global response. The probable explanation for this difference between yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage.[46]

In general global response to DNA damage involves expression of multiple genes responsible for postreplication repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations. Yeast Rev1 and human polymerase are members of [Y family translesion DNA polymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes.[38]

DNA repair rate is an important determinant of cell pathology

Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence.[13] For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice.[47] In similar manner, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan.[48] However, not every DNA repair deficiency creates exactly the predicted effects; mice deficient in the NER pathway exhibited shortened life span without correspondingly higher rates of mutation.[49]

If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging,[13] increased sensitivity to carcinogens, and correspondingly increased cancer risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.[50]

Most life span influencing genes affect the rate of DNA damage

A number of individual genes have been identified as influencing variations in life span within a population of organisms. The effects of these genes is strongly dependent on the environment, in particular, on the organisms diet. Caloric restriction reproducibly results in extended lifespan in a variety of organisms, likely via nutrient sensing pathways and decreased metabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see[51] for some discussion); however, the behavior of many genes known to be involved in DNA repair is altered under conditions of caloric restriction.

For example, increasing the gene dosage of the gene SIR-2, which regulates DNA packaging in the nematode worm Caenorhabditis elegans, can significantly extend lifespan.[52] The mammalian homolog of SIR-2 is known to induce downstream DNA repair factors involved in NHEJ, an activity that is especially promoted under conditions of caloric restriction.[53] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents,[54] although similar effects have not been observed in mitochondrial DNA.[55]

It is interesting to note that the C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under conditions of caloric restriction.[56] This observation supports the pleiotropy theory of the biological origins of aging, which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a corresponding disadvantage late in life.

Defects in the NER mechanism are responsible for several genetic disorders, including:

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

All of the above diseases are often called segmental progerias (accelerated aging diseases) because their victims appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the symptoms of old age.

Other diseases associated with reduced DNA repair function include Fanconi anemia, hereditary breast cancer and hereditary colon cancer.

Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer.[57][58] There are at least 34 Inherited human DNA repair gene mutations that increase cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCA1 and BRCA2, two famous genes whose mutations confer a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as progenitor cells in the gut, skin, and hematopoietic system are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body).

Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations.[59]

Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification,[60] changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1)[61] and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations).

While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers.[62][63][64][65]

Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair[66][67] or in homologous recombinational repair (HRR).[68] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.[69]

Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing.[70][71]

Deficient expression of DNA repair proteins due to an inherited mutation can cause increased risk of cancer. Individuals with an inherited impairment in any of 34 DNA repair genes (see article DNA repair-deficiency disorder) have an increased risk of cancer, with some defects causing up to a 100% lifetime chance of cancer (e.g. p53 mutations).[72] However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.[73]

Deficiencies in DNA repair enzymes are occasionally caused by a newly arising somatic mutation in a DNA repair gene, but are much more frequently caused by epigenetic alterations that reduce or silence expression of DNA repair genes. For example, when 113 colorectal cancers were examined in sequence, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration).[74] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.[75][76][77][78][79]

Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1).[80] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.[81]

In further examples (tabulated in Cancer epigenetics), epigenetic defects were found at frequencies of between 13%-100% for the DNA repair genes BRCA1, WRN, FANCB, FANCF, MGMT, MLH1, MSH2, MSH4, ERCC1, XPF, NEIL1 and ATM. These epigenetic defects occurred in various cancers (e.g. breast, ovarian, colorectal and head and neck). Two or three deficiencies in the expression of ERCC1, XPF or PMS2 occur simultaneously in the majority of the 49 colon cancers evaluated by Facista et al.[82]

The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes.[83] Of these, 83 are directly employed in the 5 types of DNA repair processes illustrated in the chart. The more well studied genes central to these repair processes are also shown in the chart. As indicated by the DNA repair genes shown in red, many of the genes in these repair pathways are regulated by epigenetic mechanisms, and these are frequently reduced or silent in various cancers (marked by an asterisk). Two review articles,[65][84] and two broad experimental survey articles[85][86] document most of these epigenetic DNA repair deficiencies.

It appears that epigenetic repression of DNA repair genes in accurate DNA repair pathways are central to carcinogenesis. However microhomology-mediated end joining (MMEJ) is an additional error-prone repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5 25 complementary base pairs on both strands is identified and used as a basis to align the strands, but with mismatched ends. MMEJ removes extra nucleotides (flaps) where strands are joined, then ligates the strands to create an intact DNA double helix. MMEJ always involves at least a small deletion, so that it is a mutagenic pathway.[30]FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast,[87] prostate,[88] stomach,[89][90] neuroblastomas,[91] pancreatic,[92] and lung.[93] Other genes in the MMEJ pathway are also over-expressed in a number of cancers (see MMEJ for summary), and are shown in cyan (blue) in the chart in this section.

The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophage (viruses that infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.[94] The ability of a large number of protein structural motifs to catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see.[95]

The fossil record indicates that single-cell life began to proliferate on the planet at some point during the Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic form have been inherited by all extant life forms from their common ancestor. The emergence of Earths oxygen-rich atmosphere (known as the oxygen catastrophe) due to photosynthetic organisms, as well as the presence of potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced by oxidative stress.

On some occasions, DNA damage is not repaired, or is repaired by an error-prone mechanism that results in a change from the original sequence. When this occurs, mutations may propagate into the genomes of the cells progeny. Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential to be passed on to the organisms offspring. The rate of evolution in a particular species (or, in a particular gene) is a function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an influence over the process of evolutionary change.[96] Since the normal adaptation of populations of organisms to changing circumstances (for instance the adaptation of the beaks of a population of finches to the changing presence of hard seeds or insects) proceeds by gene regulation and the recombination and selection of gene variations alleles and not by passing on irreparable DNA damages to the offspring,[97] DNA damage protection and repair does not influence the rate of adaptation by gene regulation and by recombination and selection of alleles. On the other hand, DNA damage repair and protection does influence the rate of accumulation of irreparable, advantageous, code expanding, inheritable mutations, and slows down the evolutionary mechanism for expansion of the genome of organisms with new functionalities. The tension between evolvability and mutation repair and protection needs further investigation.

A technology named clustered regularly interspaced short palindromic repeat shortened to CRISPR-Cas9 was discovered in 2012. The new technology allows anyone with molecular biology training to alter the genes of any species with precision.[98]

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iPS cell technologies: significance and applications to …

In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinsons disease and Alzheimers disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.

The 2012 Nobel Prize in Physiology or Medicine was awarded for The discovery that mature cells can be reprogrammed to become pluripotent. First, we would like to consider the significance of this research. The lives of mammals, including humans, begin with the fertilization of an egg by a sperm cell. In humans, a blastocyst composed of 70-100 cells forms by approximately 5.5 days after fertilization. The blastocyst is composed of the inner cell mass, the cell population that has the ability to differentiate into the various cells that constitute the body (pluripotency), and the trophoblast, the cells that develop into the placenta and extra-embryonic tissues and do not contribute cells to the body. In the embryonic stage, the pluripotent cells of the inner cell mass differentiate into the three germ layers, endoderm, mesoderm, and ectoderm, which will form specific organs and tissues containing somatic stem cells with limited differentiation potencies. These somatic stem cells continue to divide and differentiate, and, by adulthood, an individual composed of 60 trillion cells is produced. Somatic stem cells born in the fetal period actively divide, and are involved in the formation and growth of various organs. However, even in the adult, somatic stem cells persist in niches in every organ and tissue, and play an important role in maintaining organ and tissue homeostasis. When cells in the inner cell mass are removed at the blastocyst stage and cultured in vitro, pluripotent embryonic stem (ES) cells are obtained. Thus, in the normal process of development, cell differentiation of the three germ layers proceeds from the simple stages of the fertilized egg and blastocyst, and ultimately produces an individual consisting of a complex cellular society.

In 1893, August Weismann argued that only germ cells (eggs and spermatozoa) maintain a determinant, which was described as heritable information essential to decide on the functions and features of all somatic cells in the body [1]. In his germ plasm theory, the determinants are lost or irreversibly inactivated in differentiated somatic cells.

It took more than 50 years for researchers to rewrite this dogma. In 1962, Sir John Gurdon demonstrated the acquisition of pluripotency by reprogramming cells to their initial stage using a novel research technique, i.e., producing cloned individuals by transferring somatic cell nuclei into eggs [2]. However, for many years, that result was regarded as a special case limited to frogs alone. The production of Dolly the sheep by transferring the nucleus of a somatic cell (mammary gland epithelial cell) by Sir Ian Wilmut in the late 1990s [3] showed that cloning could also be applied to mammals.

These brilliant previous works led to our studies that culminated in the induction of pluripotency in mouse somatic cells in 2006, using retroviral vectors to introduce four genes that encode transcription factors i.e., Oct4, Sox2, Klf4, and c-Myc. We designated these cells as iPS cells [4]. In 2007, we succeeded in generating human iPS cells using genes encoding the same four transcription factors [1]. The results of this research showed that although the developmental process was thought to be irreversible, by introducing key genes into differentiated adult cells the cells could be reset to a state in the extremely early stage of development in which they possessed pluripotency. That is, the results demonstrated that the differentiation process was reversible. This startling discovery made it necessary to rewrite the embryology textbooks.

Three major lines of research led us to the production of iPS cells [

] (Figure

). The first, as described above, was nuclear reprogramming initiated by Sir John Gurdon in his research of cloning frogs by nuclear transfer in 1962 [

] and by Sir Ian Wilmut, who cloned a mammal for the first time in 1997 [

]. In addition, Takashi Tada showed that mouse ES cells contain factors that induce reprogramming in 2001 [

]. The second line of research was factor-mediated cell fate conversion, initiated by Harold Weintraub, who showed that fibroblasts can be converted into the muscle lineage by transduction with the

gene, which encodes a muscle lineage-specific basic helix-loop-helix transcription factor in 1987 [

]. The third line of research was the development of mouse ES cells, initiated by Sir Martin Evans and Gail Martin in 1981 [

,

]. Austin Smith established culture conditions for mouse ES cells and identified many factors essential for pluripotency including leukemia inhibitory factor (LIF) in 1988 [

]. Later, he developed the method to induce the ground state of mouse ES cell self-renewal using inhibitors for mitogen-activated protein kinase and glycogen synthase kinase 3 [

], which supports the establishment of fully reprogrammed mouse iPS cells. Furthermore, James Thomson generated human ES cells [

] and established their optimal culture conditions using fibroblast growth factor-2 (FGF-2). Without these previous studies, we could never have generated iPS cells. Interest rapidly escalated, and, in tandem with the birth of iPS cell technology, pluripotency leapt into the mainstream of life sciences research in the form of reprogramming technology [

]. However, there remain many unanswered questions regarding reprogramming technology. What are the reprogramming factors in the egg cytoplasm that are active in cloning technology? What do they have in common with the factors required to establish iPS cells and what are the differences? What kind of epigenetic changes occur in association with the reprogramming?

The history of investigations of cellular reprogramming that led to the development of iPS cells. Our generation of iPS cells in 2006 [4] became possible due to three scientific lines of investigation: 1) nuclear reprogramming, 2) factor-mediated cell fate conversion, and 3) ES cells. See the text for details (modified from Reference [5] with permission).

Apart from basic research in embryology, broad interest has been drawn to the following possible applications of iPS cell research: (1) regenerative medicine, including elucidating disease pathologies and drug discovery research using iPS cell disease models, and (2) medical treatments (Figure

). In this review, we describe these potential applications in the context of the results of our own research.

The application of iPS cell technologies to medical science. iPS cell technologies can be used for medical science including 1) cell therapies and 2) disease modeling or drug development. See the text for details.

iPS cells can be prepared from patients themselves and therefore great expectations have been placed on iPS cell technology because regenerative medicine can be implemented in the form of autografts presumably without any graft rejection reactions. Although there have been some controversies [

], the immunogenicity of terminally differentiated cells derived from iPS cells can be negligible [

]. Moreover, there has been substantial interest in the possibility of regenerative medicine without using the patients own cells; that is, using iPS cell stocks that have been established from donor somatic cells that are homozygous at the three major human leukocyte antigen (HLA) gene loci and match the patients HLA type [

]. The development of regenerative medicine using iPS cells is being pursued in Japan and the USA for the treatment of patients with retinal diseases, including age-related macular degeneration [

], spinal cord injuries [

], Parkinsons disease (PD) [

,

], corneal diseases [

], myocardial infarction [

,

], diseases that cause thrombocytopenia, including aplastic anemia and leukemia [

,

], as well as diseases such as multiple sclerosis (MS) and recessive dystrophic epidermolysis bullosa [

] (Table

).

Planned clinical trials of iPS cell-based therapies

Masayo Takahashi, (RIKEN)

Retinal Pigment Epithelium (sheet)

Age-related macular degeneration (wet type)

Alfred Lane, Anthony Oro, Marius Wernig (Stanford University)

Keratinocytes

Recessive dystrophic epidermolysis bullosa (RDEB)

Mahendra Rao (NIH)

DA neurons

Parkinsons disease

Koji Eto (Kyoto University)

Megakaryocyte

Thrombocytopenia

Jun Takahashi (Kyoto University)

DA neurons

Parkinsons disease

Steve Goldman, (University of Rochester)

Oligodendrocyte precursor cell

Multiple Sclerosis

Hideyuki Okano, Masaya Nakamura (Keio University)

Neural stem/progenitor cells

Spinal Cord Injury

Shigeto Shimmura (Keio University)

Corneal endothelial cells

Corneal endothelial dysfunction

Koji Nishida (Osaka University)

Corneal epithelial cells (sheet)

Corneal epithelial dysfunction and trauma (e.g. StevensJohnson syndrome)

Yoshiki Sawa (Osaka University)

Cardiomyocytes (sheet)

Heart Failure

Keiichi Fukuda (Keio University)

Cardiomyocytes (sphere)

Heart Failure

Yoshiki Sasai and Masayo Takahashi (RIKEN)

Neuroretinal sheet including photoreceptor cells

Retinitis pigmentosa

Advanced Cell Technology

Megakaryocytes

Refractory thrombocytopenia

In 1998, Hideyuki Okano, in collaboration with Steven Goldman, demonstrated for the first time the presence of neural stem/progenitor cells (NS/PCs) in the adult human brain using a neural stem cell marker, the ribonucleic acid (RNA)-binding protein Musashi1 [30, 31]. Research on nerve regeneration then commenced in earnest. That same year, we began regenerative medicine research on neural stem cell transplantation in a rat model of SCI, and have since made progress in developing NS/PC transplantation therapies in experiments on animal models of SCI. First, motor function was restored by transplanting rat fetal central nervous system (CNS)-derived NS/PCs into a rat SCI model [32]. The same study also showed that the sub-acute phase is the optimal time for NS/PC transplantation after SCI. In this study, at least part of the putative mechanism by which NS/PC transplantation restored function was identified in animal models of SCI. Both the cell autonomous effect (such as synaptogenesis between graft-derived neurons and host-derived neurons) and non-cell autonomous (trophic) effects mediated cytokines released from the graft-derived cells are likely contributing to tissue repair and functional recovery. Subsequently, a non-human primate SCI model was developed using the common marmoset, and motor function in that model was restored by transplanting human fetal CNS-derived stem cells [33]. In the same study, a behavioral assay for motor function associated with SCI was developed. Based on these studies, a preclinical research system for cell transplantation therapy was established in a non-human primate SCI model.

Given these findings, we began preparations for clinical studies of human fetal CNS-derived NS/PC transplantation to treat SCI patients. However, with the guidelines for clinical research on human stem cells of the Japanese Ministry of Health, Labor and Welfare that came into effect in 2006, human fetus-derived cells and ES cells became ineligible for use in regenerative medicine. Thus, we had no choice but to change our strategy (human ES cells became eligible for use in the 2013 guidelines). In 2006, one of our research groups (Yamanakas group) established iPS cells from adult mouse skin cells. Hypothesizing that it might be possible to induce NS/PCs from iPS cells, we (Okanos group) turned our attention to iPS cells as a means of obtaining NS/PCs without using fetal or ES cells. Based on conditions that were developed for experiments on mouse ES cells [34, 35], NS/PCs were induced from mouse iPS cells [36]. The following year, we succeeded in restoring motor function by transplanting these mouse iPS cell-derived NS/PCs into a mouse model of SCI, and reported that when good iPS cells -derived NS/PCs, which had been pre-evaluated as non-tumorigenic by the transplantation into the brains of immunocompromised mice, were used for transplantation, motor function was restored for a long period of time without tumors developing [37]. In 2011, we succeeded in restoring motor function by transplanting human iPS cell-derived stem cells into a mouse SCI model [38]. Moreover, in 2012, motor function was restored by transplanting human iPS (line 201B7) cell-derived NS/PCs into the marmoset SCI model, and long-term motor function was recovered without observable tumor formation [39]. This finding was of great significance in terms of preclinical research, and provided a proof of concept that could potentially lead to a treatment method.

Collectively, when mouse or human iPS cells were induced to form NS/PCs and were transplanted into mouse or non-human primate SCI models, long-term restoration of motor function was induced, without tumorigenicity, by selecting a suitable iPS cell line [17, 40]. Considering the sub-acute phase (2-4 weeks after the injury) as the optimal time for iPS cells-derived NS/PCs transplantation for SCI patients, there are following major difficulties with autograft-based cell therapy. First, it takes about a few months to establish iPS cells. Second, it also takes three months to induce them into NS/PCs in vitro. Third, one more year would be required for the quality control including their tumorigenesis.

Considering these, our collaborative team (Okano and Yamanaka laboratories) are currently planning iPS-based cell therapy for SCI patients in the sub-acute phase using clinical-grade integration-free human iPS cell lines that will be generated by Kyoto Universitys Center for iPS Cell Research and Application (CiRA). We will establish a production method, as well as a storage and management system, for human iPS cell-derived NS/PCs for use in clinical research for spinal cord regeneration, build an iPS cell-derived NS/PC stock for regenerative medicine, establish safety screenings against post-transplantation neoplastic transformation, and commence clinical research (Phase IIIa) trials for the treatment of sub-acute phase SCI (Figure

). As these studies progress, the application of iPS cells to treat chronic phase SCI and stroke will be investigated. Significant therapeutic efficacy in the treatment of chronic phase SCI has not been achieved by cell transplantation alone [

]. However, clinical studies are planned using antagonists of axon growth inhibitors, such as Semaphorin3A inhibitors [

], followed by multidisciplinary rehabilitation combination therapies. We aim to perform a clinical trial based on the Pharmaceutical Affairs Act in collaboration with drug companies and to use iPS cell-derived NS/PC stocks for regenerative medicine to establish treatment methods for stroke, MS, and Huntingtons disease.

Strategies for the development of iPS cell-based cell therapy for SCI patients. Our collaborative team (Okanos group at Keio University and Yamanakas group at Kyoto University) have been developing an iPS cell-based cell therapy for SCI since 2006. Our previous preclinical studies have shown that long-term functional restoration can be obtained by transplantation of NS/PCs derived from appropriate iPS cells clones without observable tumor formation [10]. Currently, we aim to develop iPS cells-based cell therapy for SCI patients at sub-acute phase using the clinical grade iPS cell-derived NS/PCs (i.e., the role of Okanos group described in the blue box) which have been prepared from human iPS cell stock (i.e., the role of Yamanakas group described in the yellow box).

Lesion sites are difficult to access in patients with degenerative diseases of the nervous system. Therefore, in past studies, cell biological or biochemical analyses of their pathology centered on forced expression of the causative genes in non-nervous system cultured cell lines and on mice in which the causative gene was knocked out. However, in a few instances, the animal or cell models did not necessarily reflect the human pathology. Identifying cell biological or biochemical changes in the initial stages of the disease, before onset of symptoms, has been difficult given analyses conducted on postmortem brains. However, with the development of iPS cell technologies, it became possible to establish pluripotent stem cells from the somatic cells of anyone, irrespective of race, genetic background, or whether the person exhibits disease symptoms. Thus, it is no exaggeration to say that generation of disease-specific iPS cells using iPS cell technologies is the sole means of reproducing ex vivo phenomena that occur in patients in vivo, particularly for nervous system disorders. The result has been a tremendous desire by investigators who are conducting research on neurological diseases to become engaged in disease-specific iPS cell research [4345].

A variety of disease-specific iPS cells have been used to study nervous system diseases, including amyotrophic lateral sclerosis (ALS) [

], spinal muscular atrophy [

], spinobulbar muscular atrophy [

], Friedreichs ataxia [

], Alzheimers disease (AD) [

], PD [

], Huntingtons disease [

,

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The first iPS cell clinical trial insights – Stem Cell Assays

For an hour on Friday 12 September, Masayo Takahashi sat alone, calmly reflecting on the decade of research that had led up to this moment. (David Cyranoski, Nature doi:10.1038/516311a)

A year ago, the first historic transplantation of iPS cell-based product took place in Japan. Recently, the trial was halted due to RIKENs decision on changing strategy. Today, Id to summarize some new info about the first iPS cell trial, based on two recent articles.

Was treatment of the first patient safe and effective? Safe YES, as of 6 months post-transplant. One year safety report is coming soon. Some observations of potential efficacy were shared in todays issue of Cell & Gene Therapy Insights by Hardy Kagimoto (CEO of Healios KK):

She had a series of 18 anti-vascular endothelial growth factor (VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease. The results presented by Dr Takahashi showed that, after the removal of the subretinal fibrotic tissue and implantation of the iPS-cell-derived retinal pigment epithelial (RPE) cell sheet, the patient experienced no recurrence of neovascularization at the 6 month point and was free from frequent anti-VEGF injections. Her visual acuity was stabilized and there have been no safety related concerns so far.

Mutations in the product from the 2nd patient Some mutations were detected in the iPS cell-derived RPE product, prepared for the second patient. Nobody knows if these mutations were prohibitive for product release, since nobody has a guidance. What we know about these mutations:

three single-nucleotide variations (SNVs) and three copy-number variants (CNVs) were present that were not detectable in the patients original fibroblasts. The CNVs were all single-gene deletions. With regards to the SNVs, one is listed in a curated database of cancer somatic mutations, but only linked to a single cancer, reports CiRA. The mutated genes were not driver genes for tumor formation, wrote Takahashi in an e-mail.

and more from Kagimotos piece:

The result of tumorigenicity testing has proven the final RPE cells to be safe. Furthermore, the presence of genetic mutation does not necessarily mean that these RPE cells can be tumorigenic.

Regulatory change As per trial PI, Masayo Takahashi, the main reason for trial halting was a regulatory change:

Although the mutations were identified before transplanting cells into the second patient, and the mutations may have contributed to RIKENs decision not to treat, the main reason not to go ahead with the trial was because of a regulatory change, says Takahashi.

New Japanese law on Regenerative Medicine became effective after iPS trial was started and after first product was transplanted. Perhaps, the trial design was not very suitable in this new regulatory framework. But still, there is no guidance in new regulation about allowable level of mutations and methods of their detection in iPS cell products:

there is no regulation with which medical professionals are obligated to check gene modification for organ transplantation, mesenchymal stem cell injections or autologous cell therapy. The fact remains that we do not have clear guidelines today on which the whole community can reach a consensus that the second RPE cells are safe enough for implantation .

RIKENs decision It seem to me RIKENs decision on halting a trial was wise and very strategic. The first and the most important thing here:

As a pioneer of iPS cell clinical application, Riken took the responsible decision not to rush ahead with the second patients RPE cells, which could potentially damage the whole field of regenerative medicine.

The second thing is if there is no guidance on mutations, why not develop it now?

Although therefore, the cells were widely thought to be safe to use, after careful consideration, they made the decision that they would not implant another autologous cell sheet until such guidelines could be officially authorized. They are now coordinating the discussions at the Ministry of Education, Culture, Sports, Science, and Technology, and also at the Ministry of Health, Labor and Welfare to carefully discuss these issues with key opinion leaders in the field including government officials, regulatory experts, scientists and toxicologists.

The third thing is realization that auto- model is not the way go in future:

As of now, autologous would not be a feasible way of providing wide level clinical therapy, says CiRA spokesperson Peter Karagiannis. At the experimental level its fine, but if its going to be mass produced or industrialized, it has to be allogeneic.

Moving forward So, RIKEN is moving forward with allo- iPS cell-derived RPE. Moving together with CiRA. Well characterized partially matched lines with safety clearance by rigorous QC testing. However, some concerns about potential immunogenicity were expressed by peers:

But the project is fraught with uncertainty, because HLA-matched cells might still suffer immune rejection. Were not going to know until those clinical trials, says Coffey. CiRA is not typing its cells for minor histocompatibility antigens, which can cause T cellmediated transplant rejection. The current effort is going for major [histocompatibility matching], says Kapil Bharti, a stem cell researcher at the National Eye Institute.

Expectations Im positive about future iPS cell-based trials. I like the approach, which RIKEN is taking. Here some of our expectation for the future (feel free to add to the list):

PS: Emphases throughout the text are mine.

Tagged as: clinical trial, genomic stability, iPS, iPS cell bank, safety, stem cell line, Takahashi M

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The first iPS cell clinical trial insights – Stem Cell Assays

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