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Keywords:

  • human induced pluripotent stem cells;
  • transcription factors;
  • disease-specific stem cells;
  • genetic reprogramming;
  • differentiation;
  • regenerative medicine and drug screening

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease-specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy of known drugs or potential drug candidates for a particular diseased condition, limiting the need for animal studies and considerably reducing the time and money required to develop new drugs. Recently, functional neurons, cardiomyocytes, pancreatic islet cells, hepatocytes and retinal cells have been derived from human iPSCs, thus re-confirming the pluripotency and differentiation capacity of these cells. These findings further open up the possibility of using iPSCs in cell replacement therapy for various degenerative disorders. In this review we highlight the development of iPSCs by different methods, their biological characteristics and their prospective applications in regenerative medicine and drug screening. We further discuss some practical limitations pertaining to this technology and how they can be averted for the betterment of human life. Copyright © 2010 John Wiley & Sons, Ltd.


1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

The ability of a cell to give rise to all other cell types is called pluripotency. Such cells are found in blastocysts and persist briefly in embryos after implantation. The generation of pluripotent cells from differentiated adult cells has long been an important goal for scientists working in the field of progenitor cells. This objective gained even more importance when ethical and other technical concerns, such as tumor formation and immune rejection, severely restricted research with human embryonic stem cells (hESCs). Previous attempts at somatic cell nuclear transfer (cloning) and fusion of somatic cells with embryonic cells was marred by various ethical and methodological complications, which precluded their use as a routine research tool. However, it is clear that success in reprogramming adult cell lines could lead to cell lines which could emerge as excellent research tools to understand diseases and to test potential drug treatments. Also, the possibility of using cells to repair damaged organs would be available, and the cell lines would be immune to rejection as they would be derived from the patient him/herself.

The objective of directly generating pluripotent cells without using embryonic material was first achieved by Yamanaka and his team in 2006. Their approach involved the conversion of mouse skin fibroblasts to induced pluripotent stem cells (iPSCs) by overexpression of a set of key transcription factors, such as Oct4, Sox2, c-Myc and Klf4 (Takahashi and Yamanaka, 2006). This was a remarkable achievement, which stunned the research fraternity and policy makers around the world who had started showing their scepticism regarding the potential of embryonic stem cells in therapeutic applications. Later studies (Takahashi and Yamanaka, 2006; Maherali et al., 2007) proved that iPSCs shared similar characteristics to embryonic stem cells (ESCs). In 2007 (Okita et al., 2007; Takahashi et al., 2007) adult human cells were reprogrammed to generate iPSCs, thereby opening up a new chapter in the field of progenitor cell therapy.

2. Reprogramming methods and defined factors used for the production of iPSCs

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

The basic biology of iPSC development is conceptually simple, yet elegant and effective. However, the actual procedure consists of several steps, each of which is technically demanding, eventually making it tedious and requiring advanced technical skills and state-of-the-art laboratory facilities (Figure 1). Initially the reprogramming process was performed by using retroviruses to transfer 24 candidate genes into mouse fibroblasts (somatic cells). Later this number was pruned to four essential transcription factors which were found to be efficient in transforming different cell types, including those of the rhesus monkey (Yu et al., 2007) and humans (Liu et al., 2008). However one of these factors, c-Myc (a proto-oncogene) was found to induce tumours in mice and hence was excluded from the reprogramming basket, albeit at the cost of the efficiency of the process. This subtle modification has also rendered the process more time consuming, since c-Myc plays a critical role in augmenting the rate of proliferation of the somatic cells, thereby making them more receptive to reprogramming.

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Figure 1. Important steps in iPSC technology

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The transmission of these transcription factors was a carried out using nucleic acid-based delivery of the programming factors. Although initial studies used retroviral vectors, soon lentiviruses became the more popular choice because of their reduced risk. One of the primary concerns of this approach is that the viruses integrate stably and permanently within the host genome, which is not acceptable for therapeutic applications, where it is essential to have non-integrating methods. So, adenoviral delivery and transient transfection has been preferred for this purpose (Park et al., 2008). Transfection is a technique which utilizes chemical or physical methods to introduce new genes or DNA segments into cells; the introduced DNA usually does not integrate with the chromosomal DNA and hence does not affect host cell replication (Okita et al., 2008). Another group, led by Linzhao Cheng, started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPSCs from human adult as well as fetal fibroblasts. Their study showed that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23–70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1–2 weeks earlier if T was added. Upon characterization of individually picked hES-like colonies after expansion (up to 24 passages), the majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones could be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. Using this improved approach, this group also generated hES-like cells from homozygous fibroblasts containing the sickle cell anaemia mutation, Haemoglobin Sickle (Mali et al., 2008).

Current reprogramming methods to deliver reprogramming factor transgenes, including plasmid transfection, are transient and minimize the potential for the insertion of mutagenesis; nevertheless, they are currently limited by diminished reprogramming efficiencies. Therefore, of late there has been a paradigm shift in full exploration of the reprogramming strategies. piggyBac (PB) transposition is host factor-independent and has been demonstrated to be functional in various human and mouse cell lines. In 2009, Woltjen et al. (2009) demonstrated that PB transposition could efficiently reprogram fibroblasts to iPSCs. Further generation of iPSCs free of vector and transgene sequences using non-integrating episomal vectors was shown (Yu et al., 2009). However, viral and plasmid DNA incorporation into chromosomes can lead to the disruption of gene transcription and malignant transformation. Tumour formation has been found in the offspring of mice generated from blastocysts made mosaic with iPSCs. To generate iPSCs for human therapy, reprogramming should be attempted with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPSCs without viral integration, followed by successful creation of human iPSCs from embryonic fibroblasts, using adenoviral vectors expressing c-Myc, Klf4, Oct4 and Sox2 (Zhou and Freed, 2009).

To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons and recombinant proteins. Nevertheless, nucleic acid-based approaches evoke apprehension about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as providing greater control over the concentration, timing and sequence of transcription factor stimulation. In a groundbreaking report, researchers have recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogramme somatic cells into iPSCs (Zhou et al., 2009). However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion reprogramming factors in the large quantities needed for this approach using traditional heterologous in vivo production methods is difficult and cumbersome, since toxicity, product aggregation and proteolysis by endogenous proteases limit yields. Towards this goal, Yang et al. (2009) have shown that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. They used an Escherichia coli-based cell-free protein synthesis system to express a set of six human RFs, including Oct3/4, Sox2, c-Myc, Klf4, Nanog and Lin28 as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, they successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9–RF fusions. Subsequently it was shown that R9–Oct3/4, R9–Sox2 and R9–Nanog exhibit cognate DNA-binding activities, R9–Nanog translocates across the plasma and nuclear membranes, and R9–Sox2 exerts transcriptional activity on a known downstream gene target.

Forced expression of this set of pluripotent genes, by either viral or non-viral transduction, results in transgene integration with unknown and unpredictable potential mutagenic effects. Alternatively, recognition of cell culture conditions that can induce endogenous expression of these genes may be advantageous. Although primary adult human fibroblasts have basal expression of mRNA for OCT4, SOX2 and NANOG, however, translation of these messages into detectable proteins and their subcellular localization depends on cell culture conditions (Page et al., 2009). Manipulation of oxygen concentration and FGF2 supplementation can modulate the expression of some pluripotency-related genes at the transcriptional, translational and cellular localization level. In this study it has been shown that subtle changes in cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2 and NANOG, and translocation of these transcription factors to the cell nucleus (Page et al., 2009). These results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation. But this short-term induction may not be sufficient for the acquisition of true pluripotency. Further investigation of the factors involved in inducing this response could lead to discovery of defined culture conditions capable of altering cell fate in vitro. This would alleviate the need for forced expression by transgenesis, thus eliminating the risk of mutagenic effects due to genetic manipulation.

3. What is the best cellular source for iPSC technology?

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific iPSCs for disease modelling and cellular replacement therapies (Table 1). However, the low efficiency and safety issues associated with generation of human iPSCs have raised significant concerns about their prospective usage in clinical settings. Since the cell types can significantly influence reprogramming efficiency and kinetics (NIH, 2006), this topic is debatable and has gained tremendous attention of late. To date, human iPSCs have been obtained from only a few cell types. Among them, fibroblasts and keratinocytes have been the cell type of choice in the first reprogramming efforts, both in mouse and humans, because of their wide availability, easy isolation and stable genetic characteristics. Since neural cells are similar to fibroblasts in terms of their common ectodermal origin, it was hypothesized that they may be equally amenable to reprogramming by the same combination of factors. Eminli et al. (2008) first showed that neural progenitor cells (NPCs) can give rise to iPSCs. This suggested that in vitro reprogramming is a universal process. Congruent to this report, it was further demonstrated that the transcription factor OCT4 is sufficient to reprogramme human neural stem cells to pluripotency (Kim et al., 2009); these one-factor human NiPSCs resemble hESCs in global gene expression profiles, epigenetic status and pluripotency in vitro and in vivo.

Table 1. Chronological timetable of major developments in the field of iPSCs
Year of discoveryTitle of the article published in Pubmed/MedlineReference
2006Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factorsTakahashi and Yamanaka, Cell126: 663–676
2007Generation of germline-competent induced pluripotent stem cellsOkita et al., Nature448: 313–317
2007Induction of pluripotent stem cells from adult human fibroblasts by defined factorsTakahashi et al., Cell131: 861–872
2007Induced pluripotent stem cell lines derived from human somatic cellsYu et al., Science318: 1917–1920
2008Generation of mouse induced pluripotent stem cells without viral vectorsOkita et al., Science322: 949–953
2008Disease-specific induced pluripotent stem cellsPark et al., Cell134: 877–886
2008Induced pluripotent stem cells generated without viral integrationStadtfeld et al., Science322: 945–949
2008Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compoundsShi et al., Cell Stem Cell3: 568–574
2009Generation of induced pluripotent stem cells using recombinant proteinsZhou et al., Cell Stem Cell4: 381–384
2009Human induced pluripotent stem cells free of vector and transgene sequencesYu et al., Science324(5298): 79–801
2009Functional cardiomyocytes derived from human induced pluripotent stem cellsZhang et al., Circ Res104: e30–41
2009Induced pluripotent stem cells from a spinal muscular atrophy patientEbert et al., Nature457(7227): 277–280
2009Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cellsZhang et al., Cell Res19(4): 429–438
2009Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signaturesChin et al., Cell Stem Cell5(1): 111–123
2009Efficient generation of hepatocyte-like cells from human induced pluripotent stem cellsSong et al., Cell Res19(11): 1233–42
2009Generation of pluripotent stem cells from patients with type 1 diabetesMaehr et al., Proc Natl Acad Sci USA106(37): 15768–15773
2009Modelling pathogenesis and treatment of familial dysautomnia using patient-specific iPSCsLee et al., Nature461(7262): 402–406

More recently there have been some interesting reports underlining this aspect. In August 2009, for the first time a publication came out showing rapid and efficient generation of iPSCs from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4, SOX2, KLF4 and C-MYC (Li et al., 2009). Eight iPSC lines had been derived which could be continuously propagated in vitro, and expressed pluripotency markers, such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, retaining the normal karyotype. Transgenes were completely inactivated and the endogenous OCT4 promoter was adequately demethylated in the established iPSC lines. In addition, microarray analysis demonstrated a high correlation coefficient between hAFDC–iPSCs and hESCs, but a low correlation coefficient between hAFDCs and hAFDC–iPSCs (Li et al., 2009). In addition, it was established that both human and mouse melanocytes give rise to iPSCs at higher efficiencies than fibroblasts (Utikal et al., 2009). Interestingly skin fibroblasts, melanocytes and melanoma cells did not require ectopic Sox2 expression for conversion into iPSCs. iPSC lines from melanocytic cells also expressed pluripotency markers, formed teratomas and contributed to viable chimeric mice with germ line transmission (Utikal et al., 2009). More recently it was demonstrated that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ESC-like cells (Kunisato et al., 2009). This group isolated BM MNCs and embryonic fibroblasts (MEFs) from Oct4–EGFP transgenic mice, fused them with ESCs or infected them with retroviruses expressing Oct4, Sox2, Klf4 and c-Myc. Fused BM MNCs formed more ESC-like colonies than did MEFs. Infected BM MNCs gave rise to iPSCs, although transduction efficiencies were not high. BM-derived iPS (BM iPS) cells expressed ESC markers, formed teratomas, and contributed to chimeric mice with germline development (Utikal et al., 2009). These findings imply that BM MNCs have potential benefits to generate iPSCs which will certainly be more relevant to clinical application.

4. Disease-specific iPSCs and their potential applications

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

Utilization of ESCs obtained from human embryos that are shown to carry genetic disease by virtue of pre-implanted genetic diagnosis (PGD; genetic analysis of single blastomeres obtained by embryo biopsy) can yield stem cell lines that model single-gene disorders (Verlinsky et al., 2005); however, most diseases represent a much more complex genetic pattern. One strategy for producing autologous, patient-derived pluripotent stem cells is somatic cell nuclear transfer (SCNT). In a proof-of-principle experiment, SCNT-embryonic stem cells generated from mice with genetic immunodeficiency were used to combine gene and cell therapy to repair the genetic defect (Rideout et al., 2001). However, to date, SCNT has not proved successful in the human and, given the paucity of human oocytes, is destined to have restricted utility. Thus, iPSCs obtained from diseased individuals would be ideally placed not only to accelerate research towards an immaculate understanding of the disease mechanism, but also to facilitate the creation of cells for transplantation that may obviate the possibility of rejection. Furthermore, given the robustness of this strategy, direct reprogramming using a combination of master regulatory genes appears to be a simplistic source of patient-derived cell lines. However, the production of iPSCs in the majority of cases still involves the use of multiple viral vectors, precluding consideration of their use for human transplantation medicine, at least with the present state of knowledge.

However, iPSCs promise great utility in investigating several diseases. Murine models of acquired and congenital disease have been used to study human pathological conditions, but these models have their own limitations. Whenever there is a marked difference between murine and human pathophysiologies, disease-specific human iPSCs that are capable of differentiating into the affected tissue type are undoubtedly more relevant and can allow in-depth analysis of the human system in a tightly controlled situation both in vivo and in vitro. Hence, by fine-tuning the efficiency of reprogramming and scaling the tissue culture appropriately, we can employ this approach to screen other putative pluripotency factors, as well as libraries of cDNAs and small RNAs (microRNAs, SiRNAs, etc.). Therefore, being optimistic, we can say that it is possible in the laboratory to recreate or mimic the pathophysiology of any diseased condition under investigation. Further, small molecules and new drug candidates (synthetic and natural products) and/or combinations of these along with other technologies can be applied to study their effect on iPSCs, eventually leading to the establishment of a cell-based platform for drug development. Such unique cells are now a reality and the Harvard Stem Cell Institute has committed a core faculty to make these cell lines available to the research community (Maherali and Hochedlinger, 2008). Furthermore, Professor George Daley and his group have developed iPSCs from patients with a variety of genetic diseases with either Mendelian or complex inheritance, including adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman–Bodian–Diamond syndrome (SBDS), Gaucher's disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson's disease (PD), Huntington's disease (HD), juvenile-onset type 1 diabetes mellitus (JDM), Down's syndrome (DS)/trisomy 21, and the carrier state of Lesch–Nyhan syndrome (Park et al., 2008). Similarly, spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Although patient fibroblasts have been used extensively to study spinal muscular a trophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. In another landmark study of 2009, the generation of iPSCs from skin fibroblast samples taken from a child with spinal muscular atrophy was shown (Ebert et al., 2009). These cells were demonstrated to expand robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother.

Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic β cells. However, the cellular and molecular defects that cause the disease remain largely unknown. Therefore, pluripotent stem cells generated from patients with T1D would be very useful for understanding the disease modelling. To this end, a very recent report showing generation of iPSCs from patients with T1D (Maehr et al., 2009) provided strong support for successful creation of disease-specific iPSCs. In this study, adult fibroblasts from T1D patients were efficiently reprogrammed to iPSCs using three transcription factors, OCT4, SOX2 and KLF4. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathological human tissue formation in vitro, thereby enabling disease investigation and drug development.

A key challenge in this field is the demonstration of disease-related phenotypes and the ability to model the pathogenesis and treatment of disease in iPSCs. A breakthrough paper has recently been published in which the modelling of pathogenesis and the treatment of familial dysautonomia (FD) using patient-specific iPSCs has been achieved (Lee et al., 2009). FD is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood, owing to the lack of an appropriate model system. This report illustrated the derivation of patient-specific FD-iPSCs, and the directed differentiation into cells of all three germ layers including peripheral neurons has been shown (Lee et al., 2009). Gene expression analysis in purified FD-iPSC-derived lineages demonstrated tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. Furthermore, FD-iPSCs was used for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration (Lee et al., 2009). This study exemplifies the enormous promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

Recently, there has been a remarkable advancement in this exciting field of iPSC biology which has raised the bar of expectations for researchers in realizing the true potential of these reprogrammed cells. The University of Wisconsin Medical School group has successfully produced beating cardiomyocytes from human iPSCs (Zhang et al., 2009). In fact, this is another important step forward towards the effort of developing in vitro models that can help in designing novel treatment modalities that will impact clinical medicine. The developed cells were shown to differentiate into multiple cell types, including nodal, atrial and ventricular cardiomyocytes, which are typically found in the adult human heart. In a very recent report, this group has provided a detailed evaluation of the cardiac differentiation potential of human iPSCs. The cells effectively demonstrated all the attributes of cardiomyocytes, including gene expression pattern and sarcomeric organization, by immunochemical analysis. The cells were also investigated by sharp microelectrode recordings for functional competence. Three major types of action potential, ventricular, atrial and nodal, were obtained, thus establishing that all types of cells could be generated and that these cells were functionally active in vitro. Experiments were also conducted to determine whether the derived cardiomyocytes had their adrenergic pathways intact, by examining their response to isoprotorenol. Interestingly, the cells demonstrated an increased rate in response to the administration of isoprotorenol.

However, the authors of the aforesaid study have pointed out some potential problems that may delay the use of this break-through in actual clinical practice. The heterogeneity of the cardiac cells produced from pluripotent iPSCs is likely to cause arrhythmias if used in the left ventricle for cardiac repair. The possibility of tumorigenesis also cannot be ruled out, since a small population of undifferentiated cells, if present in the terminally differentiated contracting cardiomyocytes, may easily overwhelm the entire population of transplanted cells with a few cycles of cell division in vivo. Thus, approaches to producing homogeneous or well-characterized mixed cell preparations remain a great need. Another limitation observed with the in vitro differentiation of hESCs and iPSCs is that the myocytes, even after 2 months of standard two-dimensional (2D) tissue culture conditions, remain embryonic in phenotype based on their size, organization and electrical properties. Robust techniques to enhance the maturation of these cells, such as three-dimensional (3D) culture methodology, as well the application of electrical and/or mechanical stimulation, will likely help to address this issue. Nevertheless, in vitro pharmacological testing using human iPSC (hiPSC)-derived cardiomyocytes has recently been performed (Tanaka et al., 2009). In this study, hiPSCs were driven to differentiate into functional cardiomyocytes, which expressed cardiac markers including Nkx2.5, GATA4 and atrial natriuretic peptide. The hiPS-derived cardiomyocytes (hiPS-CMs) were analysed using a multi-electrode assay. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform in response to verapamil and quinidine; these changes were identical to those induced in native cardiomyocytes. This study confirms that hiPS-CMs represent a promising in vitro model for cardiac electrophysiological studies and drug screening. Subsequently it was observed that there was a surge of reports on the successful generation of various differentiated cell types, including neurons (Chambers et al., 2009), haematopoietic and endothelial cells (Choi et al., 2009), pancreatic insulin-producing cells (Zhang et al., 2009), hepatocyte-like cells (Song et al., 2009) and retinal cells (Meyer et al., 2009). In all of the aforesaid reports, the expression of early- and late-stage lineage- and tissue-specific gene and protein markers have been consistently shown to express in a time- and stage-dependent manner. Furthermore, the related functions associated with the particular cell type were comparable to those of the human ESC counterparts. In accordance with these findings, Lu et al. (2009) reported that iPSC-derived haematopoietic proegenitors showed the ability to repopulate sublethally irradiated NOD/SCID-IL-2 receptor γ-chain-null (NOG) mice for almost 1 year. This was the first evidence supporting the functionality of iPSCs in animal models, which is not only encouraging but indicates that the research is moving in the right direction.

The recently developed iPSC technique also provides new direction for vaccination. The recent report on cell-based vaccination using transplantation of iPSC-derived memory B cells (Li et al., 2009) is concomitant to this perception. In this study, somatic cells were induced to form iPSCs and expanded. Then the cells were genetically or chemically promoted to an immune cell fate, followed by in vitro antigen-presenting and -processing procedures to produce memory B cells that could secrete functional antibodies to different pathogens. Finally these cells were transplanted back into a human (Li et al., 2009).

Moreover, analogous to hESCs, iPSCs could be applied in toxicology and pharmacology. For instance, iPSCs differentiating in vitro may be used as an alternative system for the screening of embryotoxic and/or teratogenic substances (Caspi et al., 2008). Broadly, the fast-growing field of in vitro reprogramming augments the hope of cures for patients as well as the commercial interest of biotechnology companies. However, before iPSCs can be used in diagnostics and therapeutics, they must conform to several important criteria.

6. Challenges ahead and future directions

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

A critical requirement for realizing the therapeutic potential of pluripotent stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of hESCs and hiPSCs in scalable stirred-suspension bioreactors (SSBs) demonstrate that large-scale production of therapeutically useful cells is feasible with current state-of-the-art culture technologies (Kohoe et al., 2009). To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their derivatives are the actual products used in tissue engineering and cell therapies, the impact of bioreactor operating conditions on stem cell self-renewal and commitment should be considered. However, major challenges are presented that remain to be addressed before the mainstream use of SSBs for the large-scale culture of hiPSCs.

Another essential prerequisite for the future widespread application of iPSCs is the development of efficient cryopreservation methods to facilitate their storage and transportation. In an attempt to address this, a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27 632 has recently been reported (Mollamohammadi et al., 2009). When ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture (Mollamohammadi et al., 2009).

Banito et al. (2009) have also shown that senescence impairs successful reprogramming to pluripotent stem cells. This group identified senescence as one barrier leading to slow and stochastic reprogramming. Expression of the four reprogramming factors triggered senescence by upregulating p53, p16 (INK4a) and p21 (CIP1). Induction of DNA damage response and chromatin remodelling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improved the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPSCs.

Although iPSCs appear to be indistinguishable from ESCs, a recent study of gene expression profiles of mouse and hESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated (Chin et al., 2009). Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability (Chin et al., 2009).

Not only are integrating viruses crucial with regard to the potential induction of mutations and tumours by reprogrammed iPSCs in therapeutic applications, but undifferentiated iPSCs themselves would be tumorigenic in vivo, since it is very likely that donor cell grafts would be contaminated by pluripotent undifferentiated cells. Importantly, in both hESCs and iPSCs the genetic and epigenetic stability during expansion and long-term cultivation is decisive for further developments and potential applications. Hence, it is imperative to screen iPSCs and their differentiated derivatives during in vitro cultivation, not only for impending chromosomal aberrations (karyotype analysis) but also for invisible subtle genetic or epigenetic modifications that could be a risk for tumour formation. Moreover, various positive and negative selection techniques can be employed to eliminate any contamination of undifferentiated cells from the transplants. As proposed for ES cell derivatives, iPS-derived cells processed for transplantation must be tested for any undifferentiated cells that remain in the graft. To get rid of undifferentiated iPSCs, Oct4-positive cells can be sorted out using flow cytometry (Cantz et al., 2008). Theoretically, it is considered that these issues may be resolved, although at present such routine selection techniques are not easily available.

7. Concluding remarks

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References

Induced pluripotent stem cells (iPSCs) offer unparalleled potential for disease research, drug screening, toxicology and regenerative medicine. However, the process of reprogramming is inefficient and often incomplete, although it is encouraging that the technology to generate iPSCs is rapidly evolving and gradually overcoming some of the critical challenges associated with the first generation iPSCs. Recently, mouse and human iPSCs have been generated using adenovirus- or plasmid-mediated transfections or via transposons which obviate the potential concerns associated with viral integration of transgenes (Woltjen et al., 2009; Stadtfeld et al., 2008; Okita et al., 2008; Kaji et al., 2009). Further, small molecules have been employed to improve the efficiency of generating iPSC lines and allowed the use of only two transcription factors (Oct4/Sox2 or Oct4/Klf4) to generate iPSC lines (Huangfu et al., 2008; Shi et al., 2008). Given the pace of progress, it is expected that better techniques to generate human iPSCs will continue to expand rapidly. Thus, iPSC-derived neurons, cardiomyocytes, pancreatic islets and hepatocyte-like cells hold significant promise for research applications in understanding disease models, drug screening and as an autologous source of cells for tissue repair. To conclude, it would be prudent to move on to the recent review by Yamanaka (2009) about elite and stochastic models for induced pluripotent stem cell generation.

References

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Reprogramming methods and defined factors used for the production of iPSCs
  5. 3. What is the best cellular source for iPSC technology?
  6. 4. Disease-specific iPSCs and their potential applications
  7. 5. Differentiation of functional cell types from iPSCs, with special emphasis on cardiomyocytes
  8. 6. Challenges ahead and future directions
  9. 7. Concluding remarks
  10. References