Production Of iPSCs Research Articles

Reprogram or Reboot: Small Molecule Approaches for the Production of Induced Pluripotent Stem Cells and Direct Cell Reprogramming

Stem cell transplantation is a potential therapy for regenerative medicine, which aims to restore tissues damaged by trauma, aging, and diseases. Since its conception in the late 1990s, chemical biology has provided powerful and diverse small molecule tools for modulating stem cell function. Embryonic stem cells could be an ideal source for transplantation, but ethical concerns restrict their development for cell therapy. The seminal advance of induced pluripotent stem cell (iPSC) technology provided an attractive alternative to human embryonic stem cells. However, iPSCs are not yet considered an ideal stem cell source, due to limitations associated with the reprogramming process and their potential tumorigenic behavior. This is an area of research where chemical biology has made a significant contribution to facilitate the efficient production of high quality iPSCs and elucidate the biological mechanisms governing their phenotype. In this review, we summarize these advances and discuss the latest progress in developing small molecule modulators. Moreover, we also review a new trend in stem cell research, which is the direct reprogramming of readily accessible cell types into clinically useful cells, such as neurons and cardiac cells. This is a research area where chemical biology is making a pivotal contribution and illustrates the many advantages of using small molecules in stem cell research.

Improved Cell Therapy Protocols for Parkinson's Disease Based on Differentiation Efficiency and Safety of hESC-, hiPSC-, and Non-Human Primate iPSC-Derived Dopaminergic Neurons

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

Abstract 2642: Multiple roles of p53-related pathways in somatic cell reprogramming and stem cell differentiation.

Abstract The inactivation of p53 functions enhances the efficiency and decreases the latency of producing induced pluripotent stem cells (iPSC) in culture. The formation of iPSCs in culture starts with a rapid set of cell divisions followed by an epigenetic reprogramming of the DNA and chromatin. The mechanisms by which the p53 protein inhibits the formation of iPSCs are largely unknown. Employing a temperature sensitive mutant of the p53 (Trp53) gene, we examined the impact of the temporal expression of wild type p53 in preventing stem cell induction from somatic cells. We also explored how different p53 mutant alleles affect the reprogramming process. We found that little or no p53 activity favors the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs, but also induced newly formed stem cells to differentiate. Among p53 regulated genes, p21 (Cdkn1a), but not Puma (Bbc3) played a partial role in iPSCs formation probably by slowing cell division. Activation of p53 functions in iPS cells induced senescence and differentiation in stem cell populations. High rate of birth defects and increases in DNA methylation at the IGF2-H19 loci in female offspring of p53 knockout mice suggested that the absence of p53 may give rise to epigenetic instability in a stochastic fashion. Consistently, selected p53 missense mutations showed differential effects on the stem cell reprogramming efficiency in a c-Myc dependent manner. The absence of p53 activity and functions also contributed to an enhanced efficiency of iPSC production from cancer cells. The production of iPSCs in culture from normal and cancer cells, while different from each other in several ways, both responded to the inhibition of reprogramming by the p53 protein. Citation Format: Lan Yi, Chiwei Lu, Wenwei Hu, Arnold J. Levine. Multiple roles of p53-related pathways in somatic cell reprogramming and stem cell differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2013-2642

Pluripotent Stem Cell Derivation and Differentiation Toward Cardiac Muscle: Novel Techniques and Advances in Patent Literature

Pluripotent stem cells hold unprecedented potential for regenerative medicine, disease modeling and drug screening. Embryonic stem cells (ESCs), standard model for pluripotency studies, have been recently flanked by induced pluripotent stem cells (iPSCs). iPSCs are obtained from somatic cells via epigenetic and transcriptional reprogramming, overcoming ESC-related ethical issues and enabling the possibility of donor-matching pluripotent cell lines. Since the European Court of Justice banned patents involving embryo disaggregation to generate human ESCs, iPSCs can now fuel the willingness of European companies to invest in treatments based on stem cells. Moreover, iPSCs share many unique features of ESCs, such as unlimited self-renewal potential and broad differentiation capability, even though iPSCs seem more susceptible to genomic instability and display epigenetic biases as compared to ESCs. Both ESCs and iPSCs have been intensely investigated for cardiomyocyte production and cardiac muscle regeneration, both in human and animal models. In vitro and in vivo studies are continuously expanding and refining this field via genetic manipulation and cell conditioning, trying to achieve standard and reproducible products, eligible for clinical and biopharmaceutical scopes. This review focuses on the recently growing body of patents, concerning technical advances in production, expansion and cardiac differentiation of ESCs and iPSCs.

Nanog Is an Essential Factor for Induction of Pluripotency in Somatic Cells from Endangered Felids

Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.

The Choice of Expression Vector Promoter Is an Important Factor in the Reprogramming of Porcine Fibroblasts into Induced Pluripotent Cells

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells. By using different promoters to drive transgenic expression, we show that the efficiency of reprogramming varies with the promoter type. The cells transfected with two different vector systems under the control of doxycycline-induced tet operator (TetO) promoters failed to upregulate essential endogenous pluripotency genes and to maintain stable iPSC morphology, whereas with the Ef1a and CAG promoters the same vectors proved efficient in generating iPSC-like cells with high levels of endogenous pluripotency gene expression that could be maintained long term in vitro. Our results suggest that the choice of expression vector promoters could significantly influence the efficiency of iPSC production from porcine somatic cells.

Emerging menu options for gonadal reincarnation

Emerging menu options for gonadal reincarnation

Poly(ADP-ribose) polymerase 1 regulates nuclear reprogramming and promotes iPSC generation without c-Myc

Poly(ADP-ribose) polymerase 1 (Parp1) catalyzes poly(ADP-ribosylation) (PARylation) and induces replication networks involved in multiple nuclear events. Using mass spectrometry and Western blotting, Parp1 and PARylation activity were intensively detected in induced pluripotent stem cells (iPSCs) and embryonic stem cells, but they were lower in mouse embryonic fibroblasts (MEFs) and differentiated cells. We show that knockdown of Parp1 and pharmacological inhibition of PARylation both reduced the efficiency of iPSC generation induced by Oct4/Sox2/Klf4/c-Myc. Furthermore, Parp1 is able to replace Klf4 or c-Myc to enhance the efficiency of iPSC generation. In addition, mouse iPSCs generated from Oct4/Sox2/Parp1-overexpressing MEFs formed chimeric offspring. Notably, the endogenous Parp1 and PARylation activity was enhanced by overexpression of c-Myc and repressed by c-Myc knockdown. A chromatin immunoprecipitation assay revealed a direct interaction of c-Myc with the Parp1 promoter. PAR-resin pulldown, followed by proteomic analysis, demonstrated high levels of PARylated Chd1L, DNA ligase III, SSrp1, Xrcc-6/Ku70, and Parp2 in pluripotent cells, which decreased during the differentiation process. These data show that the activation of Parp1, partly regulated by endogenous c-Myc, effectively promotes iPSC production and helps to maintain a pluripotent state by posttranslationally modulating protein PARylation.

Use of a Synthetic Xeno-Free Culture Substrate for Induced Pluripotent Stem Cell Induction and Retinal Differentiation

The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3-4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.

285 GENERATION OF CANINE INDUCED PLURIPOTENT STEM CELLS FROM CANINE FETAL FIBROBLAST AND ADULT FIBROBLAST OF CLONED DOG

Induced pluripotent stem cells (iPSC) derived from a patient’s fibroblasts have been used as fine resources for studying disease mechanisms and therapeutic strategies. The dog is considered invaluable in human disease research because its genetic diseases are strikingly similar to those of human. Therefore, we generated cloned dogs and transgenic cloned dogs via somatic cell nuclear transfer. In this study, we tried to derive canine iPSCs from canine fibroblasts to establish a way to make iPSC from skin fibroblasts of transgenic cloned dogs. We isolated canine fetal fibroblast (FF) from normal beagles and adult skin fibroblast (ASF) from cloned beagles. Both ASF and FF were infected with all-in-one retroviral vector that delivers human reprogramming factors (Oct4, Sox2, Klf4, c-Myc). Ten to twenty-one days after infection, the colony-shaped structure was picked and plated on a mouse embryonic fibroblast (MEF) feeder layer, pretreated with mitomycin C. Then, all cells were cultured with DMEM/F12 supplemented with 20% fetal bovine serum, 5 ng mL–1 basic fibroblast growth factor (bFGF), 5 ng mL–1 LIF, 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin. Alkaline phosphatase (AP) activity and expression of Oct4, Sox2, SSEA1, and SSEA4, were observed in the cells to characterise the iPS cell colonies. In vitro differentiation of 10th-passage canine iPSC was performed through embryonic body formation. About 50 canine iPS-like colonies were formed on a 100-mm dish. As a result, the canine iPSC from FF (iPSC-FF) and canine iPSC from ASF (iPSC-ASF) showed typical colony morphology, and both stained positively for AP. The expression of pluripotency-associated transcription factors Oct4 and Sox2 was positively displayed in iPSC-FF colonies. The stem cell markers SSEA1 and SSEA4 were negative in canine iPSC-FF. The canine iPS-FF spontaneously differentiated into all 3 germ layers in vitro, showing positive expressions of βIII-tubulin (ectoderm), α-SMA (mesoderm), and GATA6 (endoderm). As for iPS-ASF, characterisation and in vitro differentiation experiment are in progress. These results show that canine iPS-FF are similar to embryonic stem cells in terms of morphology and the ability to differentiate into 3 germ layers. Although we did not demonstrate complete verification of canine iPS-ASF of the cloned dog, their morphology, AP expression, and iPS-FF generation should indicate the possibility of iPSC production in the cloned dog. In conclusion, retroviral transduction of 4 human reprogramming factors can reprogram canine fetal fibroblasts into canine iPSC. The technique of producing canine iPSC will stimulate the utilisation of transgenic cloned dogs and expand the range of human diseases or therapeutic application. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311011-05-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

Pluripotent Stem Cell Derivation and Differentiation Toward Cardiac Muscle: Novel Techniques and Advances in Patent Literature

Pluripotent stem cells hold unprecedented potential for regenerative medicine, disease modeling and drug screening. Embryonic stem cells (ESCs), standard model for pluripotency studies, have been recently flanked by induced pluripotent stem cells (iPSCs). iPSCs are obtained from somatic cells via epigenetic and transcriptional reprogramming, overcoming ESC-related ethical issues and enabling the possibility of donor-matching pluripotent cell lines. Since the European Court of Justice banned patents involving embryo disaggregation to generate human ESCs, iPSCs can now fuel the willingness of European companies to invest in treatments based on stem cells. Moreover, iPSCs share many unique features of ESCs, such as unlimited self-renewal potential and broad differentiation capability, even though iPSCs seem more susceptible to genomic instability and display epigenetic biases as compared to ESCs. Both ESCs and iPSCs have been intensely investigated for cardiomyocyte production and cardiac muscle regeneration, both in human and animal models. In vitro and in vivo studies are continuously expanding and refining this field via genetic manipulation and cell conditioning, trying to achieve standard and reproducible products, eligible for clinical and biopharmaceutical scopes. This review focuses on the recently growing body of patents, concerning technical advances in production, expansion and cardiac differentiation of ESCs and iPSCs. Keywords: Cardiac muscle, cardiomyocytes, differentiation, drug screening, ESCs, iPSCs, patent literature, regeneration

The Histone Methyltransferase Inhibitor BIX01294 Enhances the Cardiac Potential of Bone Marrow Cells

Bone marrow (BM) has long been considered a potential stem cell source for cardiac repair due to its abundance and accessibility. Although previous investigations have generated cardiomyocytes from BM, yields have been low, and far less than produced from ES or induced pluripotent stem cells (iPSCs). Since differentiation of pluripotent cells is difficult to control, we investigated whether BM cardiac competency could be enhanced without making cells pluripotent. From screens of various molecules that have been shown to assist iPSC production or maintain the ES cell phenotype, we identified the G9a histone methyltransferase inhibitor BIX01294 as a potential reprogramming agent for converting BM cells to a cardiac-competent phenotype. BM cells exposed to BIX01294 displayed significantly elevated expression of brachyury, Mesp1, and islet1, which are genes associated with embryonic cardiac progenitors. In contrast, BIX01294 treatment minimally affected ectodermal, endodermal, and pluripotency gene expression by BM cells. Expression of cardiac-associated genes Nkx2.5, GATA4, Hand1, Hand2, Tbx5, myocardin, and titin was enhanced 114, 76, 276, 46, 635, 123, and 5-fold in response to the cardiogenic stimulator Wnt11 when BM cells were pretreated with BIX01294. Immunofluorescent analysis demonstrated that BIX01294 exposure allowed for the subsequent display of various muscle proteins within the cells. The effect of BIX01294 on the BM cell phenotype and differentiation potential corresponded to an overall decrease in methylation of histone H3 at lysine9, which is the primary target of G9a histone methyltransferase. In summary, these data suggest that BIX01294 inhibition of chromatin methylation reprograms BM cells to a cardiac-competent progenitor phenotype.

Multiple Roles of p53-Related Pathways in Somatic Cell Reprogramming and Stem Cell Differentiation

The inactivation of p53 functions enhances the efficiency and decreases the latency of producing induced pluripotent stem cells (iPSC) in culture. The formation of iPSCs in culture starts with a rapid set of cell divisions followed by an epigenetic reprogramming of the DNA and chromatin. The mechanisms by which the p53 protein inhibits the formation of iPSCs are largely unknown. Using a temperature sensitive mutant of the p53 (Trp53) gene, we examined the impact of the temporal expression of wild type p53 in preventing stem cell induction from somatic cells. We also explored how different p53 mutant alleles affect the reprogramming process. We found that little or no p53 activity favors the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs, but also induced newly formed stem cells to differentiate. Among p53-regulated genes, p21 (Cdkn1a), but not Puma (Bbc3) played a partial role in iPSCs formation probably by slowing cell division. Activation of p53 functions in iPSCs induced senescence and differentiation in stem cell populations. High rate of birth defects and increases in DNA methylation at the IGF2-H19 loci in female offspring of p53 knockout mice suggested that the absence of p53 may give rise to epigenetic instability in a stochastic fashion. Consistently, selected p53 missense mutations showed differential effects on the stem cell reprogramming efficiency in a c-Myc dependent manner. The absence of p53 activity and functions also contributed to an enhanced efficiency of iPSC production from cancer cells. The production of iPSCs in culture from normal and cancer cells, although different from each other in several ways, both responded to the inhibition of reprogramming by the p53 protein.

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663-676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949-953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543-549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348-362, 2009), removal of the reprogramming vectors by 'piggyBac' transposition Woltjen et al. (Nature 458(7239):766-770, 2009); Kaji et al. (Nature 458(7239):771-775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964-977, 2009), episomal vectors Yu et al. (Science 324(5928):797-801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381-384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376-388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789-797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

Induced Pluripotency and Oncogenic Transformation Are Related Processes

Induced pluripotent stem cells (iPSCs) have the potential for creating patient-specific regenerative medicine therapies, but the links between pluripotency and tumorigenicity raise important safety concerns. More specifically, the methods employed for the production of iPSCs and oncogenic foci (OF), a form of in vitro produced tumor cells, are surprisingly similar, raising potential concerns about iPSCs. To test the hypotheses that iPSCs and OF are related cell types and, more broadly, that the induction of pluripotency and tumorigenicity are related processes, we produced iPSCs and OF in parallel from common parental fibroblasts. When we compared the transcriptomes of these iPSCs and OF to their parental fibroblasts, similar transcriptional changes were observed in both iPSCs and OF. A significant number of genes repressed during the iPSC formation were also repressed in OF, including a large cohort of differentiation-associated genes. iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts, but gene ontology analysis pointed toward monosaccharide metabolism as upregulated in both iPSCs and OF. iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways. We reprogrammed oncogenic foci (ROF) to produce iPSC-like cells, a process dependent on Nanog. However, the ROF had reduced differentiation potential compared to iPSC, suggesting that oncogenic transformation leads to cellular changes that impair complete reprogramming. Taken together, these findings support a model in which OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes.

Pathogenic Mutation of ALK2 Inhibits Induced Pluripotent Stem Cell Reprogramming and Maintenance: Mechanisms of Reprogramming and Strategy for Drug Identification

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

Differentiated cells can be reprogrammed into pluripotent stem cells, known as "induced pluripotent stem cells" (iPSCs), through the overexpression of defined transcription factors. The creation of iPSC lines has opened new avenues for patient-specific cell replacement therapies for regenerative medicine. However, the clinical utilization of iPSCs is largely impeded by two limitations. The first limitation is the low efficiency of iPSCs generation from differentiated cells. The second limitation is that many iPSC lines are not authentically pluripotent, as many cell lines inefficiently differentiate into differentiated cell types when they are tested for their ability to complement embryonic development. Thus, the "quality" of iPSCs must be increased if they are to be differentiated into specialized cell types for cell replacement therapies. Overcoming these two limitations is paramount to facilitate the widespread employment of iPSCs for therapeutic purposes. Here, we summarize recent progress made in strategies enabling the efficient production of high-quality iPSCs, including choice of reprogramming factors, choice of target cell type, and strategies to improve iPSC quality.

MiR-25 Regulates Wwp2 and Fbxw7 and Promotes Reprogramming of Mouse Fibroblast Cells to iPSCs

BackgroundmiRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs).MethodsWe screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.Results and conclusionWe found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

Establishment of LIF-Dependent Human iPS Cells Closely Related to Basic FGF-Dependent Authentic iPS Cells

Human induced pluripotent stem cells (iPSCs) can be divided into a leukemia inhibitory factor (LIF)-dependent naïve type and a basic fibroblast growth factor (bFGF)-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

Non-viral Expression of Mouse Oct4, Sox2, and Klf4 Transcription Factors Efficiently Reprograms Tadpole Muscle Fibers in Vivo

Adult mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by a limited combination of transcription factors. To date, most current iPSC generation protocols rely on viral vector usage in vitro, using cells removed from their physiological context. Such protocols are hindered by low derivation efficiency and risks associated with genome modifications of reprogrammed cells. Here, we reprogrammed cells in an in vivo context using non-viral somatic transgenesis in Xenopus tadpole tail muscle, a setting that provides long term expression of non-integrated transgenes in vivo. Expression of mouse mOct4, mSox2, and mKlf4 (OSK) led rapidly and reliably to formation of proliferating cell clusters. These clusters displayed the principal hallmarks of pluripotency: alkaline phosphatase activity, up-regulation of key epigenetic and chromatin remodeling markers, and reexpression of endogenous pluripotent markers. Furthermore, these clusters were capable of differentiating into derivatives of the three germ layers in vitro and into neurons and muscle fibers in vivo. As in situ reprogramming occurs along with muscle tissue repair, the data provide a link between these two processes and suggest that they act synergistically. Notably, every OSK injection resulted in cluster formation. We conclude that reprogramming is achievable in an anamniote model and propose that in vivo approaches could provide rapid and efficient alternative for non-viral iPSC production. The work opens new perspectives in basic stem cell research and in the longer term prospect of regenerative medicine protocols development.