Reprogramming Of Mouse Fibroblasts Research Articles

Abstract 19007: Lineage Reprogramming of Mouse Fibroblasts to Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of mouse fibroblasts to induced cardiomyocytes (iCMs). However, reprogramming to cardiac progenitor cells (CPCs), which may be more favorable for cell therapy because of their proliferative properties and multipotency, remains to be accomplished. This study aims to reprogram mouse fibroblasts to induced CPCs (iCPCs) by transcription factor (TF)-mediated reprogramming. We have cloned a library of 25 genes including cardiac-related TFs and chromatin remodeling agents into a novel, inducible piggyBac transposon vector. Fibroblasts were derived from a Nkx 2.5- YFP cardiac progenitor reporter mouse model. We first confirmed efficient transcription/translation of our piggyBac TF library. Next, as a control experiment neonatal cardiac fibroblasts were transfected with Gata4, Mef2c, Tbx5 or Gata4, Mef2c, Tbx5, Hand2 (published factors) and monitored for appearance of beating cells. Spontaneously beating, YFP- iCMs were observed only after application of doxycycline (25 days) for both combinations of factors, demonstrating a robust reprogramming system. We then transfected neonatal cardiac fibroblasts with entire library of 25 factors and observed YFP+ cells on doxycycline treatment. The TF pool was subsequently narrowed down to 11 essential factors. On transfection with 11 factors, YFP+ cells were first observed 4 days after doxycycline treatment. By day 20 these YFP+ cells developed into two-dimensional, highly proliferative YFP+ colonies of cells that lost their parental fibroblast morphology and exhibited a high nucleus to cytoplasmic ratio. Similar reprogramming was later accomplished using adult cardiac and adult lung fibroblasts. Untransfected/iPSC factors transfected control fibroblasts yielded no beating or YFP+ cells. qPCR analysis of these YFP+ colonies revealed an upregulation of CPC markers including Mesp1, Mef2c, Irx4 accompanied with down regulation of fibroblast specific genes such as Fsp1, consistent with reprogramming towards an iCPC state. These results support the ability of inducible piggBac-based TFs to reprogram fibroblasts to iCPCs. Current experiments are focused on optimizing culture conditions to maintain iCPCs in a proliferative state and defining their potency.

Nr 75] Role of TRIM28/KAP1 protein in reprogramming of mouse fibroblasts to induced pluripotent stem cells (miPSCs)

Nr 75] Role of TRIM28/KAP1 protein in reprogramming of mouse fibroblasts to induced pluripotent stem cells (miPSCs)

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Reprogramming of human fibroblasts toward a cardiac fate

Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach.

Direct Somatic Cell Reprogramming: Treatment of Cardiac Diseases

Cardiac diseases are the major causes of morbidity and mortality in the world. Cardiomyocyte death is a common consequence of many types of heart diseases and is usually irreversible. Scar tissues formed by cardiac fibroblasts serve compensatory roles for the injured heart but eventually weaken cardiac function and result in life-threatening heart failures. Unfortunately, adult human hearts have limited regenerative capacities. In the past decades, many interventional approaches have been taken in an attempt to restore functional cardiomyocytes in an injured heart. Promising advances have been made in directly reprogramming mouse fibroblasts into cardiomyocyte-like cells both in vitro and in vivo. Recently, several different methods have been reported, including the use of transcription factors and microRNAs. In addition, two in vivo studies showed heart function improvements with delivery of reprogramming factors in mouse infarcted hearts. Although many of these studies are at early preliminary stages, the plausibility of applying cardiac reprogramming on patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field. This review will discuss the history, recent advances and challenges of cellular reprogramming, specifically in the field of cardiac regeneration.

Reprogramming of mouse fibroblasts into induced pluripotent stem cells with Nanog

Oct4–Sox2–Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mESCs in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlie the mechanisms of Nanog in reprogramming process.

Generation of mouse induced pluripotent stem cells from different genetic backgrounds using Sleeping beauty transposon mediated gene transfer

Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR—outbred, (2) C57BL/6—inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers—e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells.

A fluorescent screening platform for the rapid evaluation of chemicals in cellular reprogramming

Current strategies to monitor reprogramming into induced pluripotent stem cells (iPSCs) are limited in that they rely on the recognition of advanced stage biomarkers or they involve the transduction of genetically-modified cells. These limitations are particularly problematic in high-throughput screenings where cell availability, low cost and a rapid experimental protocol are critical issues. Herein we report the application of a pluripotent stem cell fluorescent probe (i.e. CDy1) as a reporter for the rapid screening of chemicals in reprogramming iPSCs. CDy1 stains early-stage iPSCs at 7dpi as well as matured iPSCs; hence it can partially overcome the slow kinetics of the reprogramming process. As a proof of concept, we employed a CDy1-based screening in 384 well-plates to examine the effect of newly synthesized hydroxamic acid derivatives in reprogramming mouse fibroblasts transduced with Oct4, Sox2 and Klf-4 without c-Myc. One compound (1–26) was identified as a reprogramming enhancer by 2.5-fold and we confirmed that 1–26 behaves as a histone deacetylase (HDAC) inhibitor. The successful identification of novel small molecules enhancing the generation of iPSCs by means of a rapid and simple protocol demonstrates the suitability of this CDy1-based screening platform for the large scale and high-throughput evaluation of iPSC modulators.

Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts

Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant “pTAT-mcMyc” protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP+ colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.

Turning skin into dopamine neurons

Turning skin into dopamine neurons

Generation of retinal ganglion-like cells from reprogrammed mouse fibroblasts: IOVS 2010; 51(11): 5970-5978.

Generation of retinal ganglion-like cells from reprogrammed mouse fibroblasts: IOVS 2010; 51(11): 5970-5978.

Generation of retinal ganglion-like cells from reprogrammed mouse fibroblasts

Retinal neuron degeneration results in irreversible loss of vision. Neuroprotective strategies are effective for the early stage of degeneration, but cannot prevent the cell death in advanced stage. Stem cell transplantation into injured retina has potential to restore the vision and provide treatment of advanced stages of retinal degeneration.1,2 Embryonic stem (ES) cells have been identified as a viable source of retinal progenitors. The embryonic retinal stem cells/progenitors are however, not available in sufficient quantity for clinical application. These cells also possess limited self-renewal besides ethical issues because of their embryonic/ fetal origin. doi : 10.5214/ans.0972.7531.1118207

Top Advances in Functional Genomics and Translational Biology for 2010

In recent years, major advances have been made in transcriptomics, metabolomics, proteomics, and particularly population genetics. More than 850 genome-wide association studies (GWAS) were published by the end of 2010, now covering a vast number of diseases and phenotypes and often incorporating tens of thousands of subjects or, in some cases, more than 100 000 subjects, increasing statistical power to identify and replicate contributing loci. Sequencing throughput advances and decreasing costs per sample have allowed the application of genome-wide genotyping and sequencing technology to larger DNA samples as well as transcriptome and epigenome features. This year was marked not only by continuing progress with GWAS, sequencing, and copy number variation studies but also by follow-up of GWAS and other findings with functional studies that uncover new molecular pathways contributing to disease and pave the way for future clinical applications. The American Heart Association Functional Genomics and Translational Biology Interdisciplinary Council (http://www.americanheart.org/presenter.jhtml?identifier=3016539) provides a forum for a multidisciplinary group of volunteers committed to making a substantial contribution to reduce the burden of heart disease and stroke through genetics, genomics, proteomics, and metabolomics research and translational science. With input from the Early Career Committee of the Council on Functional Genomics and Translational Biology, we considered many papers and selected 10 outstanding manuscript groups (15 articles) published in 2010 that reflect these advances, and we briefly summarize each below in chronological order.

292 GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS FROM VARIOUS GENETIC BACKGROUND BY SLEEPING BEAUTY TRANSPOSON-MEDIATED GENE TRANSFER

Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).

Generation of Retinal Ganglion–like Cells from Reprogrammed Mouse Fibroblasts

Somatic cells can be reprogrammed into an embryonic stem cell-like pluripotent state by Oct-3/4, Sox2, c-Myc, and Klf4. Sox2 as an essential reprogramming factor also contributes to the development of the eye and the retina. This study was conducted to determine whether induced pluripotent stem (iPS) cells express retinal progenitor cell (RPC)-related genes and whether iPS cells can directly differentiate into retinal ganglion cells (RGCs). Mouse iPS cells were induced by the ectopically expressed four factors in tail-tip fibroblasts (TTFs). The expression of RPC-related genes in iPS cells was analyzed by RT-PCR and immunofluorescence. iPS cells were induced to differentiate into RGCs by the addition of Dkk1 + Noggin (DN) + DAPT and overexpression of Math5. iPS-derived retinal ganglion (RG)-like cells were injected into the retina, and the eyes were analyzed by immunohistochemistry. iPS cells inherently express RPC-related genes such as Pax6, Rx, Otx2, Lhx2, and Nestin. Overexpression of Math5 and addition of DN can directly differentiate iPS into retinal ganglion-like cells. These iPS-derived RG-like cells display long synapses and gene expression patterns, including Math5, Brn3b, Islet-1, and Thy1.2. Furthermore, inhibiting Hes1 by DAPT increases the expression of RGC marker genes. In addition, iPS-derived RG-like cells were able to survive but were unable to be integrated into the normal retina after transplantation. The four factor iPS cell inherently expressed RPC-related genes, and the iPS cell could be further turned into RG-like cells by the regulation of transcription factor expression. These findings demonstrate that iPS cells are valuable for regeneration research into retinal degeneration diseases.

Pluripotency and Reprogramming: Meeting Report on the Fifth International Meeting of the Stem Cell Network North Rhine Westphalia

In March 2009, the fifth International Meeting of the Stem Cell Network North Rhine Westphalia took place in Aachen, Germany. Numerous fascinating presentations about reprogramming, stem cells, and therapeutic devices were given. A number of excellent speakers from all over the world were invited to present their work. Over 20 high-profile presentations were given on 2 days under the five different topics: reprogramming, mechanisms regulating stem cells, stem cell differentiation, cancer stem cells, and therapeutic devices. Young researchers had opportunity to present their work in over 120 posters. The meeting started with the main topic: reprogramming. Austin J. Cooney, Baylor College of Medicine, Houston, USA, gave an exiting presentation about alternative pathways to maintain pluripotency. His work is focused on the regulation of Oct4 by nuclear receptors, specifically LRH-1, which regulates directly Oct4 expression and the role of canonical wnt signalling relating to b-catenin, which potentiates reprogramming (Gu et al., 2005). His group showed that ES cells deficient in LRH-1 and b-catenin lose their pluripotency faster than wild-type ES cells. The group established a model for generation of b-catenin = ES cells. In a second model, the effect of LRH-1 was analyzed and the effect of BIO, a GSK3b inhibitor, was evaluated. The results showed that wnt3a induces LRH-1 in a b-catenin-dependent manner, because b-catenin binds directly to TCF elements in the LRH-1 promotor (Lluis and Cosma, 2009; Mullen et al., 2007). Theodore Rasmussen, University of Connecticut, USA, talked about direct reprogramming of somatic cells: from ES cell fusion to iPS. Rasmussen and his working group chose the method of somatic cell nuclear transfer (SCNT) to reprogram differentiated somatic cells to a more pluripotent state. He pointed out that iPS technology is highly promising because it can yield immunocompatible cells by relatively simple, noncontroversial means. Rasmussen’s group used the superb cell biology allowed by SCNT to study rapid and dynamic chromatin remodeling events such as histone replacement, which occur within hours after nuclear transfer with kinetics similar to those of preimplantation development (Chang et al., 2005). FMR reprogramming takes longer than SCNT, but is faster than iPS. FMR is well suited for genetic analyses, because polymorphic differences between the somatic and ES cell fusion partners can be used to trace reprogrammed gene expression (Ambrosi et al., 2007). In addition, genes can be manipulated in ES cells prior to fusion to evaluate their importance for reprogramming. Together, SCNT and FMR offer unique advantages for the investigation of reprogramming mechanisms. The presentation of Sir John B. Gurdon, Cambridge, UK, a pioneer in this field, was focused on nuclear reprogramming by nuclear transfer. John Gurdon and his group try to identify the components of eggs that cause the nuclear reprogramming. One method is to transfer multiple nuclei into the germinal vesicle of growing eggs, the oocytes. These result in lots of mammalian somatic cell nuclei that can be induced to start expressing genes for pluripotency such as Oct4, Nanog, and Sox2. The researchers detect that the following steps are involved in reprogramming: the chromatin becomes highly decondensed, differentiation marks are removed, the provision of transcription factors does not appear to be necessary in reprogramming, and linker histones of the H1 class are exchanged in transplanted nuclei (Gurdon and Melton, 2008). The presentation of Huck-Hui Ng, Genome Institute of Singapore, was focused on the major role of transcriptional regulation in specifying the unique properties of embryonic stem cells. They used chromatin immunoprecipitation coupled to high-throughput sequencing technology to map the binding sites for 13 different transcription factors (Oct4, Sox2, Nanog, Smad1, Stat3, Esrrb, Tcfcp2l1, Klf4, c-Myc, n-Myc, Zfx, E2f1, and Ctcf ) in mouse ES cells (Chen et al., 2008). The data revealed hot spots for transcription factor binding. These hot spots occur in two major clusters, namely, the Oct4-centric clusters and the Myccentric clusters. Dr. Ng also presented data on a new reprogramming factor, Esrrb (Feng et al., 2009). Esrrb is a nuclear orphan receptor that works in conjunction with Oct4 and Sox2 to reprogram mouse fibroblasts to induced pluripotent stem cells. This study demonstrates that one of the core reprogramming factor Klf4, can be substituted by Esrrb. Three outstanding presentations on the mechanisms regulating the stem cell state were given in the second session.

On the Road to iPS Cell Cardiovascular Applications

See related article, pages 648–656 The ability to generate induced pluripotent stem (iPS) cells from somatic cells by the overexpression of a limited number of stem cell-related genes has generated great excitement and interest in the biomedical research community including cardiovascular researchers. The pioneering study by Yamanaka and colleagues showing that overexpression of Oct3/4 , Sox2 , Klf4 , and c-Myc could reprogram mouse fibroblasts to a pluripotent state similar to that of embryonic stem (ES) cells opened major new avenues of research.1 This epigenetic reprogramming was rapidly extrapolated to the human system using either the same combination of reprogramming factors or a slightly different combination of transgenes ( OCT4 , NANOG , SOX2 , LIN28 ).2–4 Like embryonic stem (ES) cells, iPS cells can be used for basic developmental biology research and also as a cell source to generate theoretically unlimited quantities of desired cell types such as cardiomyocytes. Such differentiated cells types can be used in a wide range of basic research studies and potentially in clinical applications, which not only include cellular therapies but also drug discovery and safety testing. One appealing aspect of human iPS cells compared to human ES cells is that they can be more readily generated without specialized expertise and access to human embryos, which also avoids the ethical challenges associated with human embryo research. Potentially the most powerful advantage of iPS cells over ES cells is that they can be generated from any patient to produce genetically identical pluripotent cells that can create human disease models or generate patient-specific cells for therapy. Already a number of iPS cell human disease models have been generated,5,6 and proof-of-principle iPS cellular therapies have been pioneered in mouse models.7–9 Despite the speed at which the iPS cell field is racing forward, we …

Induced pluripotent stem cell (iPS) technology: promises and challenges

In 2006, an article published in Cell by Shinya Yamanaka took by surprise the stem cell research community. By performing systematic retroviral transduction of factors enriched in embryonic stem (ES) cells, the authors demonstrated the reprogramming of mouse fibroblasts into an ES cell-like state. These cells, baptized iPS (induced pluripotent stem) cells, were immediately recognized as a ground-breaking discovery. Subsequently, the same authors and other groups reported a similar achievement with human fibroblasts. Two years later, the number of top quality papers on iPS is astonishing, and interest in the scientific community has risen to a fever pitch. But although iPS has the potential to revolutionize Regenerative Medicine, important questions still remain unanswered. Work from multiple laboratories worldwide including ours is focused on deciphering the molecular mechanisms of iPS, and trying to improve the technique to make it suitable for the clinic. In this review article we briefly discuss the past, present and future of iPS, with emphasis on urgent issues to be solved.

Reprogrammed Mouse Fibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic Lineages

Forced expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1-positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell-derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV-differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1-positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.