Review

Gene Therapy (2008) 15, 82–88; doi:10.1038/sj.gt.3303061; published online 15 November 2007

Scalable human ES culture for therapeutic use: propagation, differentiation, genetic modification and regulatory issues

M Rao1

1Stem Cells and Regenerative Medicine, Invitrogen Corporation, Carlsbad, CA, USA

Correspondence: Dr M Rao, Stem Cells and Regenerative Medicine, Invitrogen Corporation, 5781 Van Allen Way, Carlsbad, CA 92008, USA. E-mail: mahendra.rao@invitrogen.com

Received 8 September 2007; Revised 8 October 2007; Accepted 10 October 2007; Published online 15 November 2007.

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Abstract

Embryonic stem cells unlike most adult stem cell populations can replicate indefinitely while preserving genetic, epigenetic, mitochondrial and functional profiles. ESCs are therefore an excellent candidate cell type for providing a bank of cells for allogenic therapy and for introducing targeted genetic modifications for therapeutic intervention. This ability of prolonged self-renewal of stem cells and the unique advantages that this offers for gene therapy, discovery efforts, cell replacement, personalized medicine and other more direct applications requires the resolution of several important manufacturing, gene targeting and regulatory issues. In this review, we assess some of the advance made in developing scalable culture systems, improvement in vector design and gene insertion technology and the changing regulatory landscape.

Keywords:

Good Manufacturing Practice (GMP), ESC-like, screening, clinical grade

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Introduction

Human embryonic stem cell lines (hESCs) are overall similar possess high capacity for self-renewal, exhibit pluripotency and proliferate indefinitely in culture while maintaining epigenetic and karyotypic stability.1, 2 This ability of hESCs, coupled with the increased efficiency of new cell line generation and the overall similarity of the various lines isolated allows in principle for a virtually unlimited supply of cells for targeted cell therapy, large-scale drug discovery efforts, toxicology screening and novel ex vivo gene therapy techniques.1 The current model for using stem cells builds on the idea of a small source bank from which a qualified master bank is derived and used to produce working banks of cells. Since undifferentiated cells are not used directly in most applications, a process for specific cell type differentiation is developed. Further, as it is difficult to obtain a full conversion to any cell type, a process for selection of an appropriate phenotype also needs to be established as well (Figure 1). When ex vivo gene therapy is envisaged the process is modified to incorporate a step of gene insertion into the cells. This can be either by homologous recombination, site specific or random integration.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Flow chart of the cell culture and gene targeting process.

Full figure and legend (124K)

The extensive history of developing gene vectors and the large numbers of papers on growing and differentiating endometrial stromal cells (ESCs) into an appropriate phenotype suggest that in principle there is no major technological barrier to utilizing ESC-derived cells for therapy.3 What then are the difficulties in translating these basic research findings to the clinic? I believe that in addition to major technical challenges such as modulating the immune response to allow efficient integration of HLA-mismatched cells and developing methods of targeted insertion there are also several practical issues that include translating the progress made in the research laboratory to methodology that can be used to manufacture cells as a drug (product).

In this review, I will focus on three major issues that I believe need to be resolved before cell therapy can be envisaged. The first is developing scalable GMP'able methods of cell culture, the second are the challenges of combining cell therapy with gene therapy and the third is understanding the regulatory issues that need to be addressed.

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Scalable/GMP'able processes

Currently available ESC lines for the most part were harvested from fertilized embryo's using immunosurgery with guinea pig complement and growth conditions which include the exposure to xeno material. To date, embryonic stem cell cultures are generally expanded either by co-culture with MEF's or growth ‘MEF-conditioned’ medium on matrigel using serum or serum replacement media. The most common serum replacement media is knockout serum replacement (KSR) that is based on a proprietary formulation and hence the specific formulation is unknown. However, it is clear that this serum replacement is not xeno-free and likely contains porcine and murine components. Thus, current culture protocols require use of xenobiotic material, co-culture with fibroblasts, and addition of undefined components in the form of matrigel or KSR. Cells are generally passaged using a manual cutting procedure or enzymes, such as trypsin, dispase, collagenase or some mixture of the above.4 Harvested cells are generally frozen in cryovials in serum and dimethyl sulfoxide containing media.

Many of these steps present technical challenges to a scale-up procedure and to developing tests that will satisfy regulatory agencies that these cells can be manufactured reliably and reproducibly.5 Challenges that we have identified are summarized in Table 1. A particular challenge that I believe is very important to solve is growth in a xeno-free media. Although this is not critical as the Food and Drug Administration (FDA) has in the past approved material that contains xeno products we believe this will be important as the number of additional tests that need to be performed is a significant burden and the ability to obtain reliable and reproducible cell lots is generally difficult in a complex media with ill-defined conditions. Investigators have already noted that often multiple lots of serum need to be tested to obtain the right lot and even KSR can show variability. Several investigators have begun attempts to develop serum-free media and at least three commercial serum-free media formulations are available. Each of these formulations uses different combinations of growth factors but to date there is no universal agreement on an optimal medium for cell culture. Indeed, the international stem cell forum has embarked on a head-to-head comparison of available media to determine if any can be recommended for use by the stem cell banks or for the derivation of new cell line.6


A second major challenge is adapting ESC to suspension culture so as to take advantage of the efficiencies of bioreactor technology. A few recent reports have suggested that it may be possible to obtain clonal cultures7, 8 and to grow cells in standard bioreactors (Michal Amit-Technion and Dr A Robins-Novocell personal communication). However, I note that Geron which is likely to be the first company to take ESC-derived oligodendrocytes to the clinic has opted not to use bioreactor technology (Dr Leibkowski personal communication) indicating to this author that significant challenges remain.

Additional challenges include passaging adherent cells in closed culture systems, developing appropriate feeding and media change schedules to minimize cost and simplifying differentiation protocols (discussed below). Perhaps two aspects of this process that have not yet received adequate attention in our minds are storage and transport of cells.

In general, ESC themselves are not considered ideal cells to transplant but rather cells differentiated at some intermediate (but not final) stage of differentiation that is dictated by the particular therapeutic need is considered the target cell for transplantation therapy. Thus, in addition, to growing ESC on needs to develop differentiation protocols that allow one to direct differentiation toward the cell of choice and several protocols to differentiate ESC are being evaluated. In general, however, it has proven very difficult to obtain a homogenous population of differentiated cells. Differentiation protocol efficiencies range for 1–3% to perhaps 24–40%.9, 10 Many of the differentiation procedures are complex and involve multiple steps where cells have to either be passaged or transferred from one condition to another or the density of the culture or the substrate needs to be modified or cells need to be co-cultured with other cell types. Such culture changes while reasonably easy to implement at the small scale are extremely difficult to implement on a large scale or automate. Equally challenging is the removal of xeno material from such processes or minimizing lot-to-lot variability. For example, differentiation of oligodendrocytes from ESC requires isolation based on difficult to quantify criteria such as color of the embryoid bodies.11 Neural stem cell differentiation requires manual isolation of rosettes and dopaminergic neuron cultures require co-culture with PA-6 cells.12, 13 Similar issues have been identified for cardiomyocyte differentiation14 and pancreatic islet differentiation.15 Thus, one more major challenge facing the community is developing selection procedures.

Another challenge that will be faced will be shipping and tracking cells as they are transported from the manufacturing facility to the clinic. Frozen vial shipping at −20 or cryovial shipping in liquid nitrogen or live cell shipping in cell culture incubators all face regulatory, customs and traceability issues that to our knowledge have not been adequately resolved. Similar challenges were faced by the organ transplant community and the blood cell community and no doubt procedures can be adapted from processes developed by these groups.16 However, as with any such adaptation process there will no doubt be unique issues that must be resolved.

An equally important issue that must be considered is ensuring stability of the cells in culture and defining tests to monitor deviation from stability in large-scale cultures. This is a critical and perhaps underappreciated issue. Although ESC are far more stable than other cell populations and less prone to the acquisition of genetic, epigenetic or mitochondrial changes when compared to adult stem cells can nevertheless change when grown in suboptimal conditions. Such conditions induce selection processes that select for cells that have acquired a growth advantage,1 and stringent efforts are required to identify and eliminate such changes in a master or working bank. Equally important tests need to be devised that can evaluate cells rapidly and with fidelity.17

Some proposed solutions

It is important to emphasize that although these are enormous challenges, there are really no insurmountable barriers to reducing a laboratory protocol to a defined manufacturing process. No doubt, one will face many unexpected challenges, and developing a viable process will take time but already innovative solutions are being proposed. One example, perhaps, is the suicide gene approach to get rid of unwanted cells. A suicide gene encodes a protein able to convert a nontoxic prodrug into a toxic product. Therefore, cells expressing the suicide gene become selectively sensitive to the prodrug.18, 19 The transfer of a suicide gene into ESC via target site insertion could allow, upon administration of the prodrug, the in vivo selective elimination of ESC derivatives that differentiate inappropriately or did not respond to the environment appropriately. The suicide gene approach is probably of great importance to ESC protocols given that it is important to get rid of all or virtually all undifferentiated cells in a final lot of cells destined for clinical use as ESC when not exposed to appropriate differentiation signals can grow to form benign tumors even when not transformed.

An alternate strategy to target undifferentiated ESC cells that may escape differentiation signals in vitro or in vivo is to exploit cell surface receptors that are uniquely expressed on ESC. Antibodies to such receptors can be generated and conjugated to toxins (for example, saporin-conjugated antibodies) or may be directly toxic (for example, PODXL antibodies). Exposure of cells to such agents in vitro or in vivo will result in the selective killing of unwanted cells while preserving the utility of appropriately differentiated progeny.20

Similarly, advances have been made in testing technology, screening for epigenetic mutations, identifying key regulatory molecules as well as in bioreactor technology and storage solutions. Overall, it appears that progress in developing methodologies to grow and differentiate ESC in large-scale culture, and to develop protocols to differentiate and select ESC-derived populations of clinically relevant cell types has been rapid and perhaps even dramatic. Nevertheless, there remains much work to be done to develop appropriate screening tools and selection procedures and to develop scalable processes to manufacture, ship and store cells for clinical use.

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Combining cell therapy and gene therapy

It is important to note that the gene therapy field has faced many unique challenges in terms of developing tests to determine the number of residual viral particles, the probability of mobilization of inserted products and determining insertion sites when random methods of insertion are used. Nevertheless, much progress has been made and although there have been several unexpected setbacks and there is no commercial gene therapy product, there are several promising clinical trials that are ongoing. Perhaps, the major stumbling block currently is the recent observation from preclinical and clinical studies is that the semi-random insertion of transgenes into chromosomal DNA of hematopoietic cells may induce clonal competition, which potentially may even trigger leukemia or sarcoma. Insertional mutagenesis caused by gene vectors has thus led to major uncertainty among those developing advanced cell therapies using genetically modified cells. Nevertheless, the tools developed by the gene therapy community are likely to prove invaluable to enhance the utility of ESC and extend their range of use.21, 22

The ability to maintain dividing cells in prolonged culture allows researchers the ability to introduce genes into selected regions of the genome and select clones that carry the appropriate genetic modification at a targeted site and still retain enough cell self-renewal capability to obtain enough genetically modified cells for large-scale screening and ex vivo gene therapy applications. Perhaps, the most dramatic example of such efforts in nonhuman stem cell populations have been using homologous recombination in mouse embryonic stem cells. It has been possible to obtain truly remarkable stable modifications of the genome and to repeatedly targeting the same site using recombinase technology.

In principle, many of these strategies could be adapted to human cells and by combining stem cell culture with ex vivo gene insertion via homologous recombination one could resolve some of the problems discussed above. The ability of combining targeted gene insertion with embryonic stem cell culture also offers the potential of resolving some of the technical issues in scale-up that I have discussed gene transfer as a means of facilitating growth and isolation of genetically modified hESCs and as a mechanism for mitigating adverse effects associated with administration of hESCs or their derivatives. It is therefore somewhat of a surprise to the hESC research community that no additional genes have been successfully targeted since the initial report of Zwaka and Thomson,23 although another group also successfully targeted the hprt gene in 2004.24 Successful targeting unexpressed loci in the hESC genome or targeting both alleles of a gene have yet to be reported.

Elaborate targeting vector construction is a prerequisite for successful gene targeting in hESCs as is optimal selection conditions (both positive and negative) to identify rare homologous recombination events. To generate such targeting vectors rapidly, we believe it is important to use novel cloning techniques, for example, the ET cloning via homologous recombination. The ET cloning approach will allow for directly isolating genomic DNA from BACs or yeast artificial chromosomes containing human genome and eliminates the step of prior isolation of large DNA fragments. This is particularly important for hESCs since hESC lines have been derived from various ethnic groups and isogenic DNA is not readily available for targeting vector construction for each line.

It has been recently suggested that targeting efficiency using nonisogenic DNA may be as efficient or nearly as efficient as isogenic DNA in human cells.25 Indeed, we have recently successfully constructed a targeting vector directly from a commercial available human BAC source by ET cloning, and targeted a nonexpressed gene in undifferentiated hESCs with an efficiency of about 5% (6 out of 106 clones) of transfected cells (unpublished data). The ability to use nonisogenic DNA is important as this reduces the need to develop individual libraries or BAC's for each of the 150 or so currently reported ESC lines.

Other difficulties encountered in gene targeting of hESCs include the low efficiency in transfecting hESCs, poor cloning efficiency of hESCs and low frequency of homologous recombination. Recent advances in defining hESCs culture conditions without feeders25, 26, 27 and the report that hESCs can be efficiently cultured from single cells28 will likely increase transfection and cloning efficiency in hESCs. As the field advances, more efficient methods to introduce DNA into hESCs will likely be emerged. Toward the aim of increasing homologous recombination frequency, a remarkable technology using zinc-finger nucleases (ZFNs) has been developed and will be discussed in detail in the next paragraph.

Zinc-finger nucleases

Strategies to address higher efficiency of homologous recombination in mammalian cells have been proposed, and notably, the ZFN technology.29, 30 In this system, a DNA double-strand break (DSB) is introduced at a targeted chromosomal locus by engineered ZFNs that recognize DNA sequences with high specificity. Homologous recombination is then achieved by DSB-induced homology-directed DNA repair with extrachromosomal donor DNA sequences carrying the designed alternation. Because homology-directed DNA repair is a natural process for repairing DNA DSB in cells, remarkable frequency of homologous recombination (up to 18%) without any selection has been achieved in multiple cell types by this system.29 More remarkably, about 50% of the targeted cells are homozygous, even without selection in such settings.29, 30 While these results are exciting, it is important to note that so far success in identifying specific zinc fingers for all target sites remains low and many of the successful results have been obtained in cell lines and not in ESC cultures. Clearly, much work remains to be done before this process can be considered routine.

Integrase technology

Recently, the Streptomyces bacteriophage ΦC31 integrase has been reported to mediate site-specific recombination in both human and mouse cells.31 The advantage of this integrase is that it catalyzes a recombination reaction between two heterotypic sites (attP and attB) and the product sites (attL and attR) of the recombination reaction are not the substrates for subsequent recombination. Thus, the recombinant event by ΦC31 integrase is irreversible which would be useful for inserting circular DNA into the genome. Although not widely used in engineering mouse genome, the ΦC31 integrase has been demonstrated to be feasible in mESCs.31 In this setting, a DNA sequence flanked by two attP (or attB) sites was first placed into the genome of mESCs, ΦC31-mediated recombination was then occurred when incoming sequence flanked by two attB (or attP) sites in either a circular or linearized plasmid was introduced into the cells. Importantly, ΦC31-mediated recombination in mESCs did not interfere with germline transmission in mice.

It would be useful to generate a hESC line with the attP site inserted into a specific locus that does not result in gene silencing. Such a target hESC line could be used as a base for further integrations of gene expression constructs with multiple att sites so that multiple genes may be studied in the same expression context, eliminating position effects. Indeed, Invitrogen has recent tested this concept using two integrases, ΦC31 and R4, in a karyotypic abnormal hESC variant line. Unlike loxP and frt sites which do not occur in mammalian genome, pseudo att sites were identified in mouse and human genome.32 Using the pseudo attP sites a R4 site was introduced in the genome of hESCs by ΦC31 integrase. Once such a targeted hESC line was established and tested for pluripotency and gene silencing, it was used to facilitate subsequent genetic modification experiments (retargeting) by inducing a subsequent integration into the target locus in a platform line using R4 integrase (Jon Chesnut, Invitrogen, personal communication).

As with ZFNs this integrase technology is not without its own potential problems. Integrases can themselves induce chromosomal rearrangements and indeed this has been reported.33 Thus, integrase technology needs to be empirically proven in ESC and careful analysis of the genome is required to demonstrate that the cells retain a normal karyotype and single nucleotide polymorphism profile.

Another strategy that is being explored is using transposases. Transposons are mobile genetic elements that can be used for mutagenesis in a number of organisms. Recent reports showing that the Sleeping Beauty transposable system can be used for stable gene expression in the cells of vertebrates including humans suggest that transposons may be applicable to hESCs and other stem cells as well.34, 35, 36 More recently another transposase system has been described. Another modified transposon system that may be useful for targeted genetic engineering is the PiggyBac transposon system, which has recently been shown to mediate gene transfer in human cells with high efficiency without overproduction inhibition.37

Overall these results raise the possibility that the techniques that have been so successfully used in mice or novel technologies specifically developed for human cells could be used for hESC barring unexpected surprises. Early results from some groups indeed have suggested that the basic process can be adapted to human cells and that these techniques work. In addition, many ESC investigators4 have shown that sufficient numbers of cells can be obtained using available large-scale manufacturing techniques to generate sufficient numbers of cells for genetic manipulation and subsequent screening and therapeutic applications while retaining genetic and epigenetic stability. Further, appropriate drug resistant feeders, feeder-free conditions and vectors that target human ESC have been developed and tested. Although the initial reports of efficiency are low and there is no demonstration of targeting to a nonexpressed gene locus and there are reports of a higher rate of silencing than that observed in mouse it has nevertheless been possible to obtain targeted genetic modification in human ESC. Overall when one examines progress in this critical transitional area I believe one can only be favorably impressed by the progress to date while cautioned by the challenges ahead.

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Regulatory issues

Since 1998, the FDA of the United States published the rules and regulations about human somatic cell therapy and gene therapy (download from: http://www.fda.gov/cber/gdlns/somgene.pdf). This document provides detailed information regarding to the sources, types, culture procedure and quality of cells, including developing measures of cell identity and heterogeneity. All clinical research involving drugs, devices and biological products is regulated by FDA, under guidance and rules governing investigational new drugs (INDs), new biologics (Biological Licensure applications (BLA's)) or devices, regardless of the source of support. In 2004, the FDA further clarified its rules by defining which subsections of the tissue and cell procurement act would govern the use of more than minimally manipulated cells.38, 39 Although these rules have helped clarify many of the issues investigators face as they proceed toward developing cell products for therapy. Some of these issues are summarized in Table 2 and discussed briefly below.


How does one define bioequivalence

An important perhaps overlooked issue is the fact that unlike most drugs where we have good control of the source materials and manufacturing process and chemical formulations cells are intrinsically more variable. Cells change in culture over multiple passages, cells from different individuals even if identical by a variety of criteria may behave differently (for example, degree of proliferation of the same cell may be different in young and old) and as such their functional activity will be different. Equally important for field is the fact that lot sizes for treatment may be quite small. At one extreme multiple lots may be required to treat one patient (pancreatic islets for example) and this raises the question of how one defines equivalence among lots and obtains agreement on dosing. Researchers and regulatory agencies are grappling with the idea and to our knowledge there is no clear solution.

Animal models differ from humans in unpredictable ways

In a review by Ginis and Rao,40 differences between human and rodent signaling, intrinsic species differences that will affect stem cell transplantation differences in transplant surgery in humans and animal models and the overall differences between animal models and human disease were noted and their effect on developing preclinical safety data was discussed. For example, the size of rodent brain (a favored small animal model) is much smaller and the structure is much simpler than human's. The developmental processes do not match well and as such equivalent cells are difficult to identify, further given the mismatch in the equivalent development stages for each organ or each structure it will be difficult to obtain definitive data on any cell therapy. Clearly these differences mean that there is no optimal solution.

A second equally major challenge facing developing adequate data in animal models is the issue of a xenograft. Cells selected for transplant therapy irrespective of their embryonic, fetal or adult origin carry antigens on their surface that will be recognized as foreign in a HLA-mismatched transplant (or will modify the immune response in specific ways) and as with any tissue or organ transplant will either be rejected or achieve a homeostasis with the host immune system. Testing and predicting what will happen is however a major challenge for researchers. Placing human cells into an animal model requires immunosuppression or using animals which do not mount an immune response to a foreign antigen (SCID-NOD, RAG1/2 knockouts and so on) and as such the immune interactions cannot be reliably evaluated. Options include parallel tests of animal cells of the same species after transplant into the same model or more detailed testing in healthy human volunteers and neither is completely satisfactory.

How long should tumorigenicity assays last and is the current methodology reasonable?

This same issue of a xenomodel in a species that may have fundamental biochemical or physiological differences also affects assessing tumorigenicity assays that will be required. Is testing in immunosuppressed animals a reasonable strategy? Does the test have to be done in normal animals or in diseased animals and at the site of transplant or in a more standard locale? More importantly how long should one monitor the animals? Given the expectation that at least some stem cells will be curative and be persistent in the host for the host's lifetime, should tumorigenicity assays be more than the usual 90 days currently recommended? Clearly this will be an important issue that will need to be resolved before cell therapy becomes routine.

What constitutes an unacceptable genetic change?

Another important issue that cell therapy advocates face is attempting to define what is normal. Cells in culture or in vivo as people age undergo change and accumulate changes in mitochondria, methylation profiles and other epigenetic changes and may also accumulate genomic changes.41, 42 Most of these are likely of little consequence but in a small fraction the changes may be far more significant such as loss of a tumor suppressor, activation of an oncogene and so on The question for researchers and regulatory authorities is how does one distinguish trivial changes from these more serious alterations and what tests are available and even feasible. Clearly there is no consensus within the community. At one extreme, one could logically argue that an autologous transplant with minimally manipulated cells does not require any testing and this is ‘the normal’ cell for the individual. Others have argued that even if one does not manipulate the cells, the fact that we infuse more or infuse in an atypical environment changes the dynamics much as a normal cell present in an ectopic environment may cause problems and as such detailed testing should be considered. Irrespective of what the final consensus turns out to be, it is clear from this research that consensus is important particularly for the regulatory agencies.

Overall, we feel that these issues require resolution and that the matter has reached some urgency as there has been a 10-fold increase in the number of clinical trials using non-HSC cells for therapy and several IND's and BLA's are under consideration.

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Challenges ahead

As one surveys the stem cell landscape, one sees some stem cells or progenitor cells that are already in therapy, one sees some cells that are in early clinical studies and somewhere there is a great promise based on animal studies. To date most of the reported benefits have come from HSC and HSC-related products. It appears to us that the next cell type that may prove usable for therapy is the adult-derived MSC where several companies and investigators have reported utility either as an adjuvant to HSC to improve homing, as a source of cells for replacing defective cells in osteogenesis imperfecta, or as a source of trophic molecules in a variety of injuries in a variety of tissues. NSCs and progenitor cells are being explored for use in lysosomal storage disorders and treatment of PD, spinal cord injury and stroke.

Developing ESC cell therapy will take additional time and several challenges lie ahead. I believe however that several therapeutic targets cannot be served by adult stem cell populations and will require the successful development of ESC-related technology. Success in the ESC field will come in measured paces and no doubt there will be failures along the way. We will learn from them however, and I believe that there is a high likelihood of success. Developing scalable manufacturing processes, highly controlled genetic modification utilizing homologous and site-specific recombination and a sustained focus on solving the regulatory barriers will allow researchers to surmount the admittedly much higher barriers that allogeneic ESC cell therapy proponents face in moving this class of stem cell to the clinic.

Perhaps ESC proponents should also evaluate recent results showing that pluripotency can be conferred by expressing a small set of regulatory genes in somatic cells43, 44 as also recent reports of germ cell-derived pluripotent lines.45, 46 These cells can be derived from a patient and thus would be personalized ESC-like cells which perhaps could be manufactured using the techniques developed for ESC work (see NIH executive document-Plans for implementation of Executive Order 13435). These cells would offer the advantage of being isogenic and thus not prone to immune system rejection. As with any new technology, challenges remain particularly for transdifferentiated pluripotent cells. The process is currently inefficient, their remains the issue of tumorigenicity, and the regulatory hurdles faced by the multiple random insertions of genes into the genome still need to be resolved as will developing techniques to shut off or completely silence pluripotency genes when cells are differentiated to ensure the safe use of differentiated cells.

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