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Induce Pluripotent Stem Cell Methods, Development and Advances Niketan Deshmukh1 and Reshma Talkal2 ABSTRACT- The self renewal and Pluripotency of Embryonic stem cell can be used in different medical purposes. But due to ethical issues we cannot use them up to the mark. However there is a good alternative of embryonic stem cell namely induced Pluripotent stem cells (iPSCs). The generation of iPSCs from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a Pluripotent state by the enforced expression of a few embryonic transcription factors. In addition, iPSC technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders In this review, we summarize the progress that has been made in the iPSC field.

Key words: Self-renewal, Pluripotency, Embryonic stem cell, iPSCs.

INTRODUCTION Embryonic stem cells offer hope for new therapies, but their use in research has been hotly debated.

Different countries have chosen to regulate embryonic stem cell research in very different ways.

Mention embryonic stem cells in the pub and the topic still divides opinion. Embryonic stem cell research poses a moral dilemma. It forces us to choose between two moral principles: •

The duty to prevent or alleviate suffering

•

The duty to respect the value of human life

In the case of embryonic stem cell research, it is impossible to respect both moral principles. To obtain embryonic stem cells, the early embryo has to be destroyed. This means destroying a potential human life. However this limitation can be successfully overcome with the help Induced Pluripotent Stem Cell. Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, Author: Niketan Deshmukh, Mtech (Biotech), Department of Biotechnology,Lovely Professional University Phagwara 144401, India.

Email: niketan@hotmail.co.in Co-Author: Reshma Talkal, Department Biotechnology, Sindhu Mahavidyalaya Center Biotech, Nagpur 440005 India.

of of

DNA methylation patterns, doubling time, embryoid body formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed. So iPSCs represent a powerful tool for biomedical research and may provide source for replacement therapies.

Are iPSCs truly Equivalent to ESCs? ESCs and iPSCs are created using different strategies and conditions, leading researchers to ask whether the cell types are truly equivalent. To assess this issue, investigators have begun extensive comparisons to determine pluripotency, gene expression, and function of differentiated cell derivatives. Ultimately, the two cell types exhibit some differences, yet they are remarkably similar in many key aspects that could impact their application to regenerative medicine. Future experiments will determine the clinical significance (if any) of the observed differences between the cell types.

Other than their derivation from adult tissues, iPSCs meet the defining criteria for ESCs. Mouse and human iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cell types from all three primitive embryonic layers, and displaying the capacity to contribute to many different tissues when injected into mouse embryos at a very early stage of development. Initially, it was unclear that iPSCs were truly pluripotent, as early iPSC lines contributed to

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mouse embryonic development but failed to produce live-born progeny as do ESCs. In late 2009, however, several research groups reported mouse iPSC lines that are capable of producing live births,1,2 noting that the cells maintain a pluripotent potential that is "very close to" that of ESCs.2 Therefore, iPSCs appear to be truly pluripotent, although they are less efficient than ESCs with respect to differentiating into all cell types.2 In addition, the two cell types appear to have similar defense mechanisms to thwart the production of DNA-damaging reactive oxygen species, thereby conferring the cells with comparable capabilities to maintain genomic integrity.3 Undifferentiated iPSCs appear molecularly indistinguishable from ESCs. However, comparative genomic analyses reveal differences between the two cell types. For example, hundreds of genes are differentially expressed in ESCs and iPSCs,4 and there appear to be subtle but detectable differences in epigenetic methylation between the two cell types.5,6 Genomic differences are to be expected; it has been reported that gene-expression profiles of iPSCs and ESCs from the same species differ no more than observed variability among individual ESC lines.7 It should be noted that the functional implications of these findings are presently unknown, and observed differences may ultimately prove functionally inconsequential.8 Recently, some of the researchers who first generated human iPSCs compared the ability of iPSCs and human ESCs to differentiate into neural cells (e.g., neurons and glia).9 Their results demonstrated that both cell types follow the same steps and time course during differentiation.

However, although human ESCs differentiate into neural cells with a similar efficiency regardless of the cell line used, iPSC-derived neural cells demonstrate lower efficiency and greater variability when differentiating into neural cells. These observations occurred regardless of which of several iPSCgeneration protocols were used to reprogram the original cell to the pluripotent state. Experimental evidence suggests that individual iPSC lines may be "epigenetically unique" and predisposed to generate cells of a particular lineage. However, the authors believe that improvements to the culturing techniques

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may be able to overcome the variability and inefficiency described in this report.

These findings underpin the importance of understanding the inherent variability among discrete cell populations, whether they are iPSCs or ESCs. Characterizing the variability among iPSC lines will be crucial to apply the cells clinically. Indeed, the factors that make each iPSC line unique may also delay the cells' widespread use, as differences among the cell lines will affect comparisons and potentially influence their clinical behavior. For example, successfully modeling disease requires being able to identify the cellular differences between patients and controls that lead to dysfunction. These differences must be framed in the context of the biologic variability inherent in a given patient population. If iPSC lines are to be used to model disease or screen candidate drugs, then variability among lines must be minimized and characterized fully so that researchers can understand how their observed results match to the biology of the disease being studied. As such, standardized assays and methods will become increasingly important for the clinical application of iPSCs, and controls must be developed that account for variability among the iPSCs and their derivatives.

Additionally, researchers must understand the factors that initiate reprogramming towards pluripotency in different cell types. A recent report has identified one factor that initiates reprogramming in human fibroblasts,10 setting the groundwork for developing predictive models to identify those cells that will become iPSCs. An iPSC may carry a genetic "memory" of the cell type that it once was, and this "memory" will likely influence its ability to be reprogrammed. Understanding how this memory varies among different cell types and tissues will be necessary to reprogram successfully.

Principle of Reprogramming Somatic Cell It is found that the difference between in a somatic cell and embryonic stem cell is present at genomic level. Expression of some specific genes is responsible for maintained the property of self renewal and pluripotency in embryonic stem cell. But when the cell move from uncommitted stage to committed stage i.e. differentiation, the expression of these genes is stop. The cloning of Dolly

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demonstrated that nuclei from mammalian differentiated cells can be reprogrammed to an undifferentiated state by trans-acting factors present in the oocyte11, and this discovery led to a search for factors that could mediate similar reprogramming without somatic cell.

dysregulated growth in the absence of a defensive immune system. Optimization of bioengineered stem cells will likely produce specialized properties that improve stress tolerance, streamline differentiation capacity, and increase engraftment/survival to improve regenerative potential.

Recently, four transcription factors (Oct4, Sox2, cmyc, and Klf4) were shown to be sufficient to reprogram mouse fibroblasts to undifferentiated, Pluripotent stem cells (termed induced pluripotent stem (iPS) cells) 12–15. The process of reprogramming requires controlled, stoichiometric expression of transgenes for a transient period of time.16-20 .

Multiple sources of tissue such as ordinary fibroblasts,21 keratinocytes,22 hematopoietic lineages,23 or adipose tissue24 have been successfully reprogrammed. Ectopic stemness factors are sufficient to induce telomere elongation,25 histone modifications,26 secondary gene expression profiles,27 and cellular metamorphosis that collectively reestablish a self stabilizing phenotype.28 Reprogramming occurs typically within weeks, following exposure to trans-acting factors that can be delivered to the nucleus either by plasmids, viruses, or bioengineered proteins. Thus, transgene expression initiates a sequence of reprogramming events that eventually transforms a small fraction of cells (< 0.5%) to acquire an imposed pluripotent state characterized by a stable epigenetic environment indistinguishable from the blastocyst–derived natural stem cell milieu. The converted pluripotent ground state results in the maintenance of the unique developmental potential with the ability to differentiate into all germ layers.

METHODS OF IPSC PRODUCTION

Induced pluripotent stem cell platforms bypass the need for embryo extraction to generate genuine pluripotent stem cells from self-derived, autologous sources. In the mouse, bioengineering strategies have yielded iPS cells sufficient for complete de novo embryogenesis as the highest evidence of pluripotent stringency.33,34 In humans29,30,31,32, iPS cells have ensured comprehensive multi-lineage tissue differentiation by demonstrating the ability to give rise to all three germ layers in teratoma formation.

Self-derived iPS cells are recognized within the transplanted hosts as native tissue due to their autologous status and thus require protection from

Retroviral metod of iPSC Production In the first works on murine and human iPSC production, either retro– or lentiviral vectors were used for the delivery of Oct4 , Sox2 , Klf4 , and c–Myc genes into somatic cells. The efficiency of transduction with retroviruses is high enough, although it is not the same for different cell types.

Retroviral integration into the host genome requires a comparatively high division rate, which is characteristic of the relatively narrow spectrum of cultured cells. Moreover, the transcription of retroviral construct under the control of a promoter localized in 5’LTR (long terminal repeat) is terminated when the somatic cell transform switches to the pluripotent state 36. This feature makes retroviruses attractive in iPSC production.

Nevertheless, retroviruses possess some properties that make iPSCs that are produced using them improper for cell therapy of human diseases. First, retroviral DNA is integrated into the host cell genome. The integration occurs randomly; i.e., there are no specific sequences or apparent logic for retroviral integration. The copy number of the exogenous retroviral DNA that is integrated into a genome may vary to a great extent 35. Retroviruses being integrated into the cell genome can introduce promoter elements and polyadenylation signals; they can also interpose coding sequences, thus affecting transcription. Second, since the transcription level of exogenous Oct4 , Sox2 ,Klf4 , and c–Myc in the retroviral construct decreases with cell transition into the pluripotent state, this can result in a decrease in the efficiency of the stable iPSC line production, because the switch from the exogenous expression of pluripotency genes to their endogenous expression may not occur. Third, some studies show that the transcription of transgenes can resume in the cells derived from iPSCs 36. The high probability that the ectopic Oct4 , Sox2 , Klf4 , and c–Myc gene

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expression will resume makes it impossible to apply iPSCs produced with the use of retroviruses in clinical trials; moreover, these iPSCs are hardly applicable even for fundamental studies on reprogramming and pluripotency principles.

Lentiviruses used for iPSC production can also be integrated into the genome and maintain their transcriptional activity in pluripotent cells. One way to avoid this situation is to use promoters controlled by exogenous substances added to the culture medium, such as tetracycline and doxycycline, which allows the transgene transcription to be regulated. iPSCs are already being produced using such systems 37 .

Another serious problem is the gene set itself that is used for the induction of pluripotency 38.

The ectopic transcription of Oct4 , Sox2 , Klf4 , and c–Myc can lead to neoplastic development from cells derived from iPSCs, because the expression of Oct4 , Sox2 , Klf4, and c–Myc genes is associated with the development of multiple tumors known in oncogenetics . In particular, the overexpression ofOct4 causes murine epithelial cell dysplasia 39, the aberrant expression of Sox2 causes the development of serrated polyps and mucinous colon carcinomas 40, breast tumors are characterized by elevated expression of Klf4 [2 , and the improper expression of c–Myc is observed in 70% of human cancers 41.

Tumor development is oberved in ~50% of murine chimeras obtained through the injection of retroviral iPSCs into blastocysts, which is very likely associated with the reactivation of exogenous c– Myc 42,43.

Non-retroviral Methods of iPSC Production A huge barrier to the eventual use of iPSCderived treatments is the use of retroviruses to force the expression of the four key genes, discussed above, and activating their transcription factors.

Retroviruses can carry target DNA that is inserted into a host cell’s genome upon injection, making them ideal for incorporating the four genes into target cells. However, this DNA and the rest of the viruses’ genomes remain in the host genome, which can lead to transcription of unwanted genes and greatly increases the risk of tumors. The expression of the four transgenes must be silenced after

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reprogramming to avoid harmful gene expression. cMyc, a tumor-promoting gene, especially must be silenced after cellular reprogramming or the risk of tumor development becomes too great for clinical use. These retroviral methods in which the transgenes are still present in the pluripotent cells pose a danger to safety, and also are less closely related to ESCs in gene expression than their non-retroviral alternatives. Methods of reprogramming iPSCs without transgene expression in the reprogrammed cell are therefore essential not only for potential therapies and clinical applications, but also for reliable and accurate in vitro models of diseases. Yet, the low efficiency of alternatives remains a worry. Whether these methods will be viable for human clinical use remains to be seen.

Excisions strategy of iPSC generation The excision strategy (transient transfection) of iPSC generation allows the transgenes to briefly integrate into the genome but then removes them once reprogramming is achieved. An example of this site/enzyme combination is the loxP site and the Cre enzyme. In a study of Parkinson’s disease (PD), specific iPSCs, this loxP/Cre combination was used to generate the iPSCs. Neural differentiation was then induced on the iPSCs to test whether they could differentiate into dopaminergic neurons, the cells harmed in PD. The differentiation was successful, indicating the transgenes had been excised. However, a loxP site remains in iPSC genome as does some residual viral DNA, so there is still a small potential for insertional mutagenesis.

The piggyBac site/enzyme system on the other hand is capable of excising itself completely, not leaving any remnants of external DNA in the iPSC genome. The piggyBac system also was much more efficient than other non-retroviral methods, with comparable efficiency to retroviral methods, but with the added benefits of safety and ease of application.

Adenoviral Methods Adenoviral methods do not pose the same threats as retroviral methods of generating iPSCs. Adenoviruses work like all viruses by hijacking their hosts’ cellular machinery to replicate their own genome and reproduce, but unlike retroviruses they do not incorporate their genome into the host DNA.

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Because the transgenes are never even incorporated into the host’s genome they do not have to be excised. Instead, the genes are expressed directly from the virus’ genome. iPSCs created by adenoviral methods demonstrated pluripotency, but have extremely low reprogramming efficiency. Viral genomic material could not be detected in any of the iPSCs, and no tumor formation was reported. This suggests that the use of nonintegrating adenoviral methods substantially lowers the threat of tumorgenesis. The successful creation of iPSCs from adenoviral methods proves definitively that safer, non-retroviral methods can also successfully reengineer cells.

Non-DNA methods of iPSC generation Recent studies have implied that perhaps genetic material is not required for iPS cellular reprogramming. The substitution of transgenes with small molecules that promote iPSC generation would be a safe, clinically appropriate way of creating iPSCs, though it remains to be seen if small molecules will be able to completely replace genetic methods of iPSC generation or are just useful as supplementary aids to the process. Protein transduction is a different method shown to entirely replace gene delivery. In this method fusion proteins are created, which fuse each of the transgenes to a cell-penetrating peptide sequence that allows it to cross the cellular membrane Reprogramming without DNA intermediates should eliminate the risk of tumorgenesis and distorted gene expression due to the reactivation of the transgenes.

Methods for iPSC production modification of the cell genome.

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Cre–loxP– Mediated Recombination.

To prepare iPSCs from patients with Parkinson’s disease, lentiviruses were used, the proviruses of which can be removed from the genome by Cre – recombinase. To do this, the loxP –site was introduced into the lentiviral 3’LTR–regions containing separate reprogramming genes under the control of the doxycycline–inducible promoter.

During viral replication, loxP was duplicated in the 5’LTR of the vector. As a result, the provirus integrated into the genome was flanked with two loxP –sites. The inserts were eliminated using the temporary transfection of iPSCs with a vector expressing Cre –recombinase 44.

In another study, murine iPSCs were produced using a plasmid carrying the Oct4 , Sox2 , Klf4I, and c–Myc genes in the same reading frame in which individual cDNAs were separated by sequences encoding 2А peptides, and practically the whole construct was flanked with loxP –sites 45. The use of this vector allowed a notable decrease in the number of exogenous DNA inserts in the host cell’s genome and, hence, the simplification of their following excision 46. It has been shown using lentiviruses carrying similar polycistronic constructs that one copy of transgene providing a high expression level of the exogenous factors Oct4, Sox2, Klf4, and c–Myc is sufficient for the reprogramming of differentiated cells into the pluripotent state47,48.The drawback of the Cre–loxP – system is the incomplete excision of integrated sequences; at least theloxP –site remains in the genome, so the risk of insertion mutations remains.

without Plasmid Vectors.

As was mentioned above, for murine and human iPSC production, both retro– and lentiviruses were initially used as delivery vectors for the genes required for cell reprogramming. The main drawback of this method is the uncontrolled integration of viral DNA into the host cell’s genome. Several research groups have introduced methods for delivering “pluripotency genes” into the recipient cell which either do not integrate allogenic DNA into the host genome or eliminate exogenous genetic constructs from the genome.

The application of lentiviruses and plasmids carrying the loxP –sites required for the elimination of transgene constructs modifies, although insignificantly, the host cell’s genome. One way to avoid this is to use vector systems that generally do not provide for the integration of the whole vector or parts of it into the cell’s genome. One such system providing a temporary transfection with polycistronic plasmid vectors was used for iPSC production from mEFs. A polycistronic plasmid carrying the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as a plasmid expressing c–Myc , was transfected into

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mEFs one, three, five, and seven days after their primary seeding. Fibroblasts were passaged on the ninth day, and the iPSC colonies were selected on the 25th day. Seven out of ten experiments succeeded in producing GFP–positive colonies (the Gfp gene was under the control of the Nanog gene promoter). The iPSCs that were obtained were similar in their features to murine ESCs and did not contain inserts of the used DNA constructs in their genomes.

Therefore, it was shown that wholesome murine iPSCs that do not carry transgenes can be reproducibly produced, and that the temporary overexpression ofOct4 , Sox2 , Klf4 , and c–Myc is sufficient for reprogramming. The main drawback of this method is its low yield. In ten experiments the yield varied from 1 to 29 iPSC colonies per ten million fibroblasts, whereas up to 1,000 colonies per ten millions were obtained in the same study using retroviral constructs.

Episomal VectorsHuman iPSCs were successfully produced from skin fibroblasts using single transfection with polycistronic episomal constructs carrying various combinations of Oct4 , Sox2 ,Nanog , Klf4 , c– Myc , Lin28 , and SV40LT genes. These constructs were designed on the basis of the oriP/EBNA1 (Epstein–Barr nuclear antigen–1) vector 48. The oriP/EBNA1 vector contains the IRES2 linker sequence allowing the expression of several individual cDNAs (encoding the genes required for successful reprogramming in this case) into one polycistronic mRNA from which several proteins are translated. The oriP/EBNA1 vector is also characterized by low–copy representation in the cells of primates and can be replicated once per cell cycle (hence, it is not rapidly eliminated, the way common plasmids are). Under nonselective conditions, the plasmid is eliminated at a rate of about 5% per cell cycle 49. In this work, the broad spectrum of the reprogramming factor combinations was tested, resulting in the best reprogramming efficiency with cotransfection with three episomes containing the following gene sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + S V40LT + Klf4 , and c–Myc + Lin28 .SV40LT ( SV40 large T gene ) neutralizes the possible toxic effect of с–Мус overexpression 50. The authors have shown

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that wholesome iPSCs possessing all features of pluripotent cells can be produced following the temporary expression of a certain gene combination in human somatic cells without the integration of episomal DNA into the genome. However, as in the case when plasmid vectors are being used, this way of reprogramming is characterized by low efficiency.

In separate experiments the authors obtained from 3 to 6 stable iPSC colonies per 106 transfected fibroblasts. Despite the fact that skin fibroblasts are well–cultured and accessible, the search for other cell types which are relatively better cultured and more effectively subject themselves to reprogramming through this method is very likely required. Another drawback of the given system is that this type of episome is unequally maintained in different cell types.

PiggyBac–TranspositionOne promising system used for iPSC production without any modification of the host genome is based on DNA transposons. So– called PiggyBac –transposons containing 2А– linkered reprogramming genes localized between the 5’– and 3’–terminal repeats were used for iPSC production from fibroblasts. The integration of the given constructs into the genome occurs due to mutual transfection with a plasmid encoding transposase. Following reprogramming due to the temporary expression of transposase, the elimination of inserts from the genome took place 51,52. One advantage of the PiggyBac system on Cre–loxP is that the exogenous DNA is completely removed 53 .However, despite the relatively high efficiency of exogenous DNA excision from the genome byPiggyBac –transposition; the removal of a large number of transposon copies is hardly achievable.

Nonintegrating Viral VectorsMurine iPSCs were successfully produced from hepatocytes and fibroblasts using four adenoviral vectors nonintegrating into the genome and carrying the Oct4 , Sox2 ,Klf4 , and c–Myc genes.

An analysis of the obtained iPSCs has shown that they are similar to murine ESCs in their properties (teratoma formation, gene promoter DNA methylation, and the expression of pluripotent markers), but they do not carry insertions of viral

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DNA in their genomes 54. Later, human fibroblast– derived iPSCs were produced using this method 55.

The authors of this paper cited the postulate that the use of adenoviral vectors allows the production of iPSCs, which are suitable for use without the risk of viral or oncogenic activity. Its very low yield (0.0001–0.001%), the deceleration of reprogramming, and the probability of tetraploid cell formation are the drawbacks of the method. Not all cell types are equally sensitive to transduction with adenoviruses.

Another method of gene delivery based on viral vectors was recently employed for the production of human iPSCs. The sendai–virus (SeV)– based vector was used in this case 56. SeV is a single– stranded RNA virus which does not modify the genome of recipient cells; it seems to be a good vector for the expression of reprogramming factors.

Vectors containing either all “pluripotency factors” or three of them (without с–Мус ) were used for reprogramming the human fibroblast. The construct based on SeV is eliminated later in the course of cell proliferation. It is possible to remove cells with the integrated provirus via negative selection against the surface HN antigen exposed on the infected cells.

The authors postulate that reprogramming technology based on SeV will enable the production of clinically applicable human iPSCs 56.

Cell Transduction with Recombinant ProteinsAlthough the methods for iPSC production without gene modification of the cell’s genome (adenoviral vectors, plasmid gene transfer, etc.) are elaborated, the theoretical possibility for exogenous DNA integration into the host cell’s genome still exists. The mutagenic potential of the substances used presently for enhancing iPSC production efficiency has not been studied in detail. Fully checking iPSC genomes for exogenous DNA inserts and other mutations is a difficult task, which becomes impossible to solve in bulk culturing of multiple lines. The use of protein factors delivered into a differentiated cell instead of exogenous DNA may solve this problem. Two reports have been published to date in which murine and human iPSCs were produced using the recombinant Oct4, Sox2, Klf4, and c–Myc proteins 57,58 . T he method used to deliver

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the protein into the cell is based on the ability of peptides enriched with basic residues (such as arginine and lysine) to penetrate the cell’s membrane.

Murine iPSCs were produced using the recombinant Oct4, Sox2, Klf4, and c–Myc proteins containing eleven C–terminal arginine residues and expressed in E. coli . The authors succeeded in producing murine iPSCs during four rounds of protein transduction into embryonic fibroblasts. However, iPSCs were only produced when the cells were additionally treated with 2–propylvalerate (the deacetylase inhibitor). The same principle was used for the production of human iPSCs, but protein expression was carried out in human HEK293 cells, and the proteins were expressed with a fragment of nine arginins at the protein C–end. Researchers have succeeded in producing human iPSCs after six transduction rounds without any additional treatment.

The efficiency of producing human iPSC in this way was 0.001%, which is one order lower than the reprogramming efficiency with retroviruses. Despite some drawbacks, this method is very promising for the production of patient–specific iPSCs. Potential Medical Applications of iPSCs iPSCs have the potential to become multipurpose research and clinical tools to understand and model diseases, develop and screen candidate drugs, and deliver cell-replacement therapy to support regenerative medicine. This section will explore the possibilities and the challenges that accompany these medical applications, with the caveat that some uses are more immediate than others. For example, researchers currently use stem cells to test/screen drugs or as study material to identify molecules or genes implicated in regeneration. Conducting experiments or testing candidate drugs on human cells grown in culture enables researchers to understand fundamental principles and relationships that will ultimately inform the use of stem cells as a source of tissue for transplantation. Therefore, using iPSCs in cellreplacement therapies is a future application of these cells, albeit one that has tremendous clinical potential. The following discussion will highlight recent efforts toward this goal while recognizing the challenges that must be overcome for these cells to reach the clinic.

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Reprogramming technology offers the potential to treat many diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disease, diabetes, and amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). In theory, easily-accessible cell types (such as skin fibroblasts) could be biopsied from a patient and reprogrammed, effectively recapitulating the patient's disease in a culture dish. Such cells could then serve as the basis for autologous cell replacement therapy. Because the source cells originate within the patient, immune rejection of the differentiated derivatives would be minimized. As a result, the need for immunosuppressive drugs to accompany the cell transplant would be lessened and perhaps eliminated altogether. In addition, the reprogrammed cells could be directed to produce the cell types that are compromised or destroyed by the disease in question. A recent experiment has demonstrated the proof of principle in this regard,59 as iPSCs derived from a patient with ALS were directed to differentiate into motor neurons, which are the cells that are destroyed in the disease.

Although much additional basic research will be required before iPSCs can be applied in the clinic, these cells represent multi-purpose tools for medical research. Using the techniques described in this article, researchers are now generating myriad disease-specific iPSCs. For example, dermal fibroblasts and bone marrow-derived mesencyhmal cells have been used to establish iPSCs from patients with a variety of diseases, including ALS, adenosine deaminase deficiency-related severe combined immunodeficiency, Shwachman- Bodian-Diamond syndrome, Gaucher disease type III, Duchenne and Becker muscular dystrophies, Parkinson's disease, Huntington's disease, type 1 diabetes mellitus, Down syndrome/trisomy, and spinal muscular atrophy.60,61 iPSCs created from patients diagnosed with a specific genetically-inherited disease can then be used to model disease pathology. For example, iPSCs created from skin fibroblasts taken from a child with spinal muscular atrophy were used to generate motor neurons that showed selective deficits compared to those derived from the child's unaffected mother.62 As iPSCs illuminate the development of normal and disease-specific pathologic tissues, it is expected that discoveries made using these cells will

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inform future drug development or other therapeutic interventions.

One particularly appealing aspect of iPSCs is that, in theory, they can be directed to differentiate into a specified lineage that will support treatment or tissue regeneration. Thus, somatic cells from a patient with cardiovascular disease could be used to generate iPSCs that could then be directed to give rise to functional adult cardiac muscle cells (cardiomyocytes) that replace diseased heart tissue, and so forth. Yet while iPSCs have great potential as sources of adult mature cells, much remains to be learned about the processes by which these cells differentiate. For example, iPSCs created from human63 and murine fibroblasts64,65 can give rise to functional cardiomyocytes that display hallmark cardiac action potentials. However, the maturation process into cardiomyocytes is impaired when iPSCs are usedâ&#x20AC;&#x201D;cardiac development of iPSCs is delayed compared to that seen with cardiomyocytes derived from ESCs or fetal tissue. Furthermore, variation exists in the expression of genetic markers in the iPSC-derived cardiac cells as compared to that seen in ESC-derived cardiomyocytes. Therefore, iPSCderived cardiomyocytes demonstrate normal commitment but impaired maturation, and it is unclear whether observed defects are due to technical (e.g., incomplete reprogramming of iPSCs) or biological barriers (e.g., functional impairment due to genetic factors). Thus, before these cells can be used for therapy, it will be critical to distinguish between iPSC-specific and disease-specific phenotypes.

However, it must be noted that this emerging field is continually evolving; additional basic iPSC research will be required in parallel with the development of disease models. Although the reprogramming technology that creates iPSCs is currently imperfect, these cells will likely impact future therapy, and "imperfect" cells can illuminate many areas related to regenerative medicine.

However, iPSC-derived cells that will be used for therapy will require extensive characterization relative to what is sufficient to support disease modeling studies. To this end, researchers have begun to use imaging techniques to observe cells that are undergoing reprogramming to distinguish true iPSCs from partially-reprogrammed cells. The

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potential for tumor formation must also be addressed fully before any iPSC derivatives can be considered for applied cell therapy. Furthermore, in proposed autologous therapy applications, somatic DNA mutations (e.g., non-inherited mutations that have accumulated during the person's lifetime) retained in the iPSCs and their derivatives could potentially impact downstream cellular function or promote tumor formation (an issue that may possibly be circumvented by creating iPSCs from a "youthful" cell source such as umbilical cord blood). Whether these issues will prove consequential when weighed against the cells' therapeutic potential remains to be determined. While the promise of iPSCs is great, the current levels of understanding of the cells' biology, variability, and utility must also increase greatly before iPSCs become standard tools for regenerative medicine.

CONCLUSIONS Stem cell biology has been an area of great interest and intense debate since its inception, and iPSC technology has furthered this research and created hope for potential therapeutic applications.

While there are still many barriers to the clinical use of stem cells, iPSCs may help elucidate the nature of both pluripotent stem cells and of many disease pathologies to reach an eventual concrete connection between the two. Reprogramming adult tissues to embryonic-like states has countless prospective applications to regenerative medicine, drug development, and basic research on stem cells and developmental processes. To this point, a PubMed search conducted in April 2010 using the term "induced pluripotent stem cells" (which was coined in 2006) returned more than 1400 publications, indicating a highly active and rapidlydeveloping research field.

However, many technical and basic science issues remain before the promise offered by iPSC technology can be realized fully. For putative regenerative medicine applications, patient safety is the foremost consideration. Standardized methods must be developed to characterize iPSCs and their derivatives. Furthermore, reprogramming has demonstrated a proof of-principle, yet the process is currently too inefficient for routine clinical

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application. Thus, unraveling the molecular mechanisms that govern reprogramming is a critical first step toward standardizing protocols. A grasp on the molecular underpinnings of the process will shed light on the differences between iPSCs and ESCs (and determine whether these differences are clinically significant). Moreover, as researchers delve more deeply into this field, the effects of donor cell populations can be compared to support a given application; i.e., do muscle-derived iPSCs produce more muscle than skin-derived cells? Based on the exciting developments in this area to date, induced pluripotent stem cells will likely support future therapeutic interventions, either directly or as research tools to establish novel models for degenerative disease that will inform drug development. While much remains to be learned in the field of iPSC research, the development of reprogramming techniques represents a breakthrough that will ultimately open many new avenues of research and therapy.

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