Tail-tip Fibroblasts Research Articles

Generation of neural progenitor cells by chemical cocktails and hypoxia

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors.

Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism.

High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays. Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester. This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.

Engineering cardiac muscle: new ways to refurbish old hearts?

Myocardial infarction leads to irreversible cell loss and scar formation, which result in impaired cardiac function, cardiac hypertrophy and, eventually, heart failure. As the intrinsic regenerative capacity of the human heart is very limited, organ transplantation or ventricular assist devices remain the only end-stage therapy options to date. For at least two decades, alternative therapeutic concepts of heart repair based either on direct cell injection or on the transplantation of in vitro engineered tissue constructs have been considered potential approaches to overcome the shortage of donor organs. However, the multitude of recent experimental and clinical studies applying various adult stem cells have thus far not been able to fulfil the initial high hopes vested in adult stem cell-based therapies. As summarized in a recent meta study, the vast majority of these studies resulted either in no or in only minor functional improvement [1], most likely due to paracrine effects, including accelerated revascularization, enhanced myocyte survival in the infarct border zone and the modulation of scar formation, resulting in improved mechanical properties [2]. Despite sporadic myocyte formation, a significant generation of de novo myocardium could not be demonstrated [3]. Interestingly, very recent studies have demonstrated the possibility of targeted trans-differentiation of cardiac fibroblasts into functional cardiomyocytes (CMs). Inspired by a recent approach utilizing the transcription factor MyoD to drive transdifferentiation of fibroblasts into skeletal muscle [4], a set of cardiac transcription factors was overexpressed for the targeted differentiation of mouse cardiac and tail-tip fibroblasts (Fbs) into cardiomyocytes in vitro [5]. Although trans-differentiation of cardiac Fbs in vivo could be demonstrated [6], the efficiency of such direct conversion experiments is still controversial [7, 8]. At this point, the usefulness of this kind of cell conversion remains uncertain and will critically depend not only on a much higher efficiency, but also on the functional similarity of these artificially converted cells into normal cardiomyocytes. For many years, embryonic stem cells (ESCs) were considered the only cell source suitable to supply the huge numbers of cardiomyocytes lost after myocardial infarction. However, a potential clinical use of ESCs is highly controversial, since their generation requires the destruction of human embryos and, if not produced through therapeutic cloning, no autologous ESCs are available for cellular therapies. In 2006, these limitations were overcome by the groundbreaking development of induced pluripotent stem cells (iPSCs) by S. Yamanaka [9], who was awarded the Nobel Prize in Medicine in 2012. In the meantime, the generation of human iPSCs has become a standard procedure in many laboratories and it is now clear that these cells are almost indistinguishable from ESCs with respect to their phenotype, culture characteristics and potential for proliferation and differentiation. Unfortunately, the availability of human iPSCs and CMs derived from these cells (Figure 1) has solved only one of the most severe limitations for current concepts for myocardial repair. Further critical hurdles are low cell survival and the lack of functional integration of the cellular transplants. In particular, the functional coupling and formation of well-organized myocardium appears to be difficult to achieve. The direct injection of cells to improve heart function was investigated in different animal models with a variety of mouse and human cell sources, including undifferentiated cells as well as cells that had been differentiated to cardiac progenitors or cardiomyocytes. Regardless of the cell type or application method (intramyocardial or intracoronary injection), only up to 5% of the cells remained in the heart, and neither significant integration nor long-term survival could be shown ([10], reviewed by ref. [11]). Although survival and cardiovascular in vitro differentiation was demonstrated, the injection of iPSC-derived cardiovascular progenitors did not result in the formation of structured myocardium [12]. In contrast, a very recent study was the first to report the formation of relatively large electrically coupled and well-structured muscle islands from injected human ESC-derived myocytes in a guinea pig model of myocardial infarction [13]. These data are considered to be very promising, although it was necessary to inject extremely high numbers of myocytes, (i.e. 10 cells, which correlates to 2 × 10 or approximately four times the total number of myocytes in the adult human left ventricle, when projected from the guinea pig to the human heart, which is ca. two hundred times larger). Even more remarkable is the fact that the formation of large contractile human ESC-derived muscle islands have evidently been confirmed by the same group in a

Reprogramming- and pluripotency-associated membrane proteins in mouse stem cells revealed by label-free quantitative proteomics

Induced pluripotent stem cells (iPSCs), derived from somatic cells and functionally very similar to embryonic stem cells (ESCs), are at the center stage of intense research in regenerative medicine. We carried out the first membrane proteomic profiling of mouse iPSCs, in comparison with ESCs and adult mouse tail tip fibroblasts (TTFs) from which iPSCs were generated. Using a proteomic workflow combining membrane fractionation, SDS-PAGE separation and nanoUPLC-MS(E) technology, we identified 673, 679 and 682 non-redundant proteins from mouse iPSC, ESC and TTF membrane fractions, respectively. Label-free quantitation revealed 155 reprogramming-associated and 128 pluripotency-associated transmembrane proteins. Furthermore, a small group of 23 membrane proteins mainly involved in amino acid/glucose/ion transport, membrane fusion and vesicular trafficking were found potentially regulated between miPSCs and mESCs. Expression changes of selected proteins were verified by qPCR, western blot and/or immunofluorescence analyses in a wider array of cell types. Notably, epithelial cell adhesion molecules, glucose transporters 1 and 3, transferrin receptor and several nuclear membrane-associated components were highly expressed in both iPSCs and ESCs, relative to TTFs. Moreover, knock-down of glucose transporter 3 in ESCs impaired the beating function of ESC-derived cardiomyocytes, suggesting its potential role in mediating stem cell differentiation. This study constitutes a membrane proteomic resource for murine iPSCs and ESCs, and offers a comparison between pluripotent stem cells and fibroblasts in the proteomic landscape. An integrated proteomics platform combining technologies of membrane fractionation, LC-MS(E) analysis and label-free quantitation was developed to identify membrane proteins with their abundances related to reprogramming of fibroblasts or maintenance of stem cell pluripotency. The high similarity in the membrane proteomic patterns between iPSCs and ESCs strengthens the usefulness of iPSCs in biomedical research and therapeutic application. Moreover, we found a small subset of membrane proteins potentially regulated between miPSCs and mESCs. This membrane proteomic resource of pluripotent stem cells would be expected to inspire further investigations leading to discovery of new regulatory factors or membrane markers for reprogramming and pluripotency.

Modeling Human Protein Aggregation Cardiomyopathy Using Murine Induced Pluripotent Stem Cells

Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Cells derived from murine induced pluripotent stem cells (iPSC) by treatment with members of TGF-beta family give rise to osteoblasts differentiation and form bone in vivo

BackgroundInduced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells into embryonic like state (ESC) using defined factors. There is great interest in these cells because of their potential for application in regenerative medicine.ResultsiPSC reprogrammed from murine tail tip fibroblasts were exposed to retinoic acid alone (RA) or in combination with TGF-β1 and 3, basic fibroblast growth factor (bFGF) or bone morphogenetic protein 2 (BMP-2). The resulting cells expressed selected putative mesenchymal stem cells (MSCs) markers; differentiated toward osteoblasts and adipocytic cell lineages in vitro at varying degrees. TGF-beta1 and 3 derived-cells possessed higher potential to give rise to osteoblasts than bFGF or BMP-2 derived-cells while BMP-2 derived cells exhibited a higher potential to differentiate toward adipocytic lineage. TGF-β1 in combination with RA derived-cells seeded onto HA/TCP ceramics and implanted in mice deposited typical bone. Immunofluorescence staining for bone specific proteins in cell seeded scaffolds tissue sections confirmed differentiation of the cells into osteoblasts in vivo.ConclusionsThe results demonstrate that TGF-beta family of proteins could potentially be used to generate murine iPSC derived-cells with potential for osteoblasts differentiation and bone formation in vivo and thus for application in musculoskeletal tissue repair and regeneration.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Epigenetic modifications and mRNA levels of the imprinted gene Grb10 in serially passaged fibroblast cells

The imprinted gene Grb10 influences fetal growth, adult behavior and tumor formation. Investigating this gene often involves consecutive passaging of cells in vitro. We analyzed changes in Grb10 DNA methylation and histone modifications in serially passaged adult mouse tail-tip fibroblast cells with bisulfite genomic sequencing method and chromatin immunoprecipitation (ChIP) respectively. There was a significant reduction in global DNA methylation, but no changes in methylation at CpG island 1 (CGI1) of Grb10. There were no changes in methylation at histone H3K4me2, but less methylated histone H3K27me3 was detected, along with more of the acetylated histones H3Ac and H4Ac. The major-type transcript level of Grb10 in fibroblasts increased mildly after extended cultivation. These results suggest consecutive passaging may affect epigenetic modifications of Grb10 in adult fibroblast cells.

Induced Pluripotent Stem–Induced Cells Show Better Constitutive Heterochromatin Remodeling and Developmental Potential After Nuclear Transfer Than Their Parental Cells

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5

Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

Heart repair by reprogramming non-myocytes with cardiac transcription factors

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Fates of Murine Pluripotent Stem Cell-Derived Neural Progenitors following Transplantation into Mouse Cochleae

This study evaluated the tumorigenesis risk of induced pluripotent stem (iPS) cells after transplantation into the cochlea. One mouse embryonic stem (ES) cell line and three mouse iPS cell lines, one derived from adult mouse tail-tip fibroblasts (TTFs) and two from mouse embryonic fibroblasts (MEFs), were neurally induced by stromal cell-inducing activity. Before transplantation, the efficiency of neural induction and the proportion of residual undifferentiated cells were evaluated using immunocytochemistry, and no significant differences were observed in the ratios of colonies expressing βIII tubulin, nestin, or octamer (Oct)3/4. Four weeks after transplantation into the cochleae of neonatal mice, the number of surviving transplants of TTF-derived iPS cells generated by retroviral infection was significantly higher than those of MEF-derived iPS cells generated by plasmid transfection. Teratoma formation was identified in one of five cochleae transplanted with TTF-derived iPS cells. However, no significant differences were found in the cell proliferation activity or the extent of differentiation into mature neurons among the cell lines. These findings emphasize the necessity of selecting appropriate iPS cell lines and developing methods to eliminate undifferentiated cells after neural induction, in order to establish safe iPS cell-based therapy for the inner ear.

Generation of Melanocytes from Induced Pluripotent Stem Cells

Epidermal melanocytes play an important role in protecting skin from ultraviolet (UV) rays, and are implicated in a variety of skin diseases. Here, we developed an efficient method for differentiating induced pluripotent stem cells (iPSCs) into melanocytes. We first generated iPSCs from adult mouse tail-tip fibroblasts (TTFs) using retroviral vectors or virus-free piggyBac transposon vectors carrying murine Sox2, Oct3/4, cMyc and Klf4. The TTF-derived iPSC clones exhibited similar morphology and growth properties as mouse embryonic stem (ES) cells. The iPSCs expressed ES cell markers, displayed characteristic epigenetic changes and formed teratomas with all three germ layers. The iPSCs were used to generate embryoid bodies (EBs) and were then successfully differentiated into melanocytes by treatment with growth factors. The iPSC-derived melanocytes expressed characteristic melanocyte markers and produced melanin pigment. Electron microscopy showed that the melanocytes contained mature melanosomes. We manipulated the conditions used to differentiate iPSCs to melanocytes and discovered that Wnt3a is not required for mouse melanocyte differentiation. This report shows that melanocytes can be readily generated from iPSCs, providing a powerful resource for the in vitro study of melanocyte developmental biology and diseases. By inducing iPSCs without viruses, the possibility of integration mutagenesis is alleviated, providing iPSCs are more compatible for cell replacement therapies.

Gene-Correction Rescues Reprogramming of Fanconi Anemia Fibroblasts and Enables Hematopoietic Differentiation of FA Induced Pluripotent Stem Cells in Vitro and In Vivo

Abstract 672Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and repairing DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells but previous attempts at gene therapy failed largely due to inadequate numbers of hematopoietic stem cells available for gene correction and autologous engraftment. Induced pluripotent stem cells (iPSC) constitute an alternate source of autologous cells, which are amenable to ex vivo expansion and genetic correction. While fibroblasts from a limited number of FA patients have been reported to fail to undergo reprogramming (Raya et al., Nature, 2009), reproducible observations and mechanistic studies ascertained in an extended panel of patient cells and murine knock-out models are lacking to date. We undertook direct reprogramming of ten unique human FA primary fibroblast samples of the FA-A, FA-C, FA-G, and FA-D2 complementation groups. Using standard four-factor reprogramming, no human FA iPSC colonies were obtained in cells defective in the FA pathway. By contrast, reprogramming of gene-corrected patient samples, augmented by hypoxia (5%O2), yielded multiple pluripotent iPSC lines, confirming a critical cell-intrinsic role of the FA pathway in reprogramming. To determine if gene-corrected FA iPSC could be therapeutically useful, we performed karyotype analyses and evaluated in vitro hematopoietic differentiation in three FA-A iPSC lines. These FA patient iPSC lines were karyotypically normal and showed a robust multilineage hematopoietic differentiation potential, resulting in erythroid and myeloid hematopoietic colony forming units to a similar degree as compared to normal donor iPSC controls. We hypothesized that the reprogramming resistance of FA cells is due to defective DNA repair and genomic instability. To explore the mechanisms of the reprogramming defect, we transduced wild type (wt) tail-tip fibroblasts (TTF) with the reprogramming vectors. We observed significantly increased FANCD2 foci formation during reprogramming (median percentage of FANCD2 foci: mock-transduced TTF 2.5%, reprogrammed TTF 20.5%, n=8, p<0.01) indicating activation of the FA pathway. Next, we examined reprogramming in FA-deficient mouse cells. We observed a significantly higher incidence of reprogramming-induced double-strand DNA breaks and senescence in Fanca−/− TTF as compared to wt controls (γH2AX foci: wt 13%, Fanca−/− 19%; senescence: wt 47%, Fanca−/− 62%, median percentage, p<0.01). To evaluate whether these changes contribute to the reprogramming resistance of FA cells, we quantified the reprogramming efficiency of Fanca−/−, Fancc−/− and littermate wt TTF. The efficiency was 0.06% for Fanca−/− (n=8) and 0.38% for Fancc−/− (n=12) as compared to 0.55% for wt controls (n=13; p<0.01 and <0.05, respectively). To directly test the role of the FA pathway in reprogramming, TTF were transduced with retroviral vectors co-expressing FANCA and enhanced green fluorescent protein (eGFP) or encoding only eGFP as a control. Under hypoxic conditions, gene-correction of the Fanca−/− TTF with FANCA resulted in a significant reduction of senescence and rescued the reprogramming efficiency of Fanca−/− TTF to wt levels. While significant chromosomal aberrations were observed in uncorrected Fanca−/− iPCS, gene-corrected Fanca−/− iPSC did not show any significant chromosomal imbalances when analyzed by comparative genomic hybridization. To evaluate the capacity of FA iPSC to form blood cells in vivo, we injected wt, control transduced or gene-corrected Fanca−/− iPCS (CD45.1+) into wt blastocysts (CD45.2+) and analyzed the contribution of iPSC-derived hematopoietic cells in embryonic day 14.5 fetal livers. We observed 1.8–4% wt iPSC chimerism (n=15), 0.4–0.9% Fanca−/− iPSC chimerism (n=3) and 1.5 to 2.5% chimerism in gene-corrected Fanca−/− iPSC (n=11). Our data demonstrate that reprogramming activates the FA pathway. Gene-correction rescues the reprogramming block of FA cells and protects FA iPSC from genomic instability, thus yielding an expandable source of autologous stem cells with hematopoietic differentiation capacity that may be explored for future use in regenerative medicine. Disclosures:Daley:iPierian, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees.

Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.

Functional Integration of Dopaminergic Neurons Directly Converted from Mouse Fibroblasts

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors. However, it is not clear to what extent this type of reprogramming is able to generate fully functional differentiated cells. In addition, the activity of the reprogrammed cells in cell transplantation assays, such as those envisaged for cell-based therapy of Parkinson's disease (PD), remains to be determined. Here we show that ectopic expression of defined transcription factors in mouse tail tip fibroblasts is sufficient to induce Pitx3+ neurons that closely resemble midbrain dopaminergic (DA) neurons. In addition, transplantation of these induced DA (iDA) neurons alleviates symptoms in a mouse model of PD. Thus, iDA neurons generated from abundant somatic fibroblasts by direct lineage reprogramming hold promise for modeling neurodegenerative disease and for cell-based therapies of PD.

Generation of induced pluripotent stem (iPS) cells derived from a murine model of Pompe disease and differentiation of Pompe-iPS cells into skeletal muscle cells

Our study is the first to demonstrate the ability to generate iPS cells from a mouse model of Pompe disease. Initially, mouse tail tip fibroblasts were harvested from male, 8-week-old (GAA) knockout mice, and three reprogramming factors (Oct3/4, Sox2 and Klf4) were transfected into the isolated donor cells using a retroviral vector. These iPS cells also showed decreased levels of GAA enzymatic activity and strong positive staining with periodic acid-Schiff (indicating the accumulation of glycogen) and acid phosphatase (lysosomal activation marker). Pompe-iPS cells were differentiated into skeletal muscle cells in Matrigel®-coated plates. Spindle-shaped skeletal muscle cells were successfully generated from Pompe-iPS cells and showed spontaneous contraction and positive staining with the myosin heavy chain antibody. Electron microscopic analysis of the skeletal muscle cells showed typical morphological features, including Z-bands, I-bands, A-bands and H-bands, which were visible in wild-type and Pompe cells. Furthermore, Pompe skeletal muscle cells accumulated massive glycogen in lysosomes. This study indicates that the iPS and skeletal muscle cells generated in this study could also be a useful disease model for studies investigating the pathogenesis and treatment of skeletal muscle in Pompe disease.

Cell Fate Plug and Play: Direct Reprogramming and Induced Pluripotency

Building on the discovery that MyoD expression reprograms fibroblasts into muscle, three papers (Vierbuchen et al., 2010; Ieda et al., 2010; Szabo et al., 2010) recently reported the reprogramming of fibroblasts into neurons, cardiomyocytes, and blood cell progenitors without first passing the cells through a pluripotent state. Here we discuss the advantages and challenges of harnessing this direct reprogramming method for regenerative medicine.

Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors

The generation of functional hepatocytes independent of donor liver organs is of great therapeutic interest with regard to regenerative medicine and possible cures for liver disease. Induced hepatic differentiation has been achieved previously using embryonic stem cells or induced pluripotent stem cells. Particularly, hepatocytes generated from a patient's own induced pluripotent stem cells could theoretically avoid immunological rejection. However, the induction of hepatocytes from induced pluripotent stem cells is a complicated process that would probably be replaced with the arrival of improved technology. Overexpression of lineage-specific transcription factors directly converts terminally differentiated cells into some other lineages, including neurons, cardiomyocytes and blood progenitors; however, it remains unclear whether these lineage-converted cells could repair damaged tissues in vivo. Here we demonstrate the direct induction of functional hepatocyte-like (iHep) cells from mouse tail-tip fibroblasts by transduction of Gata4, Hnf1α and Foxa3, and inactivation of p19(Arf). iHep cells show typical epithelial morphology, express hepatic genes and acquire hepatocyte functions. Notably, transplanted iHep cells repopulate the livers of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice and rescue almost half of recipients from death by restoring liver functions. Our study provides a novel strategy to generate functional hepatocyte-like cells for the purpose of liver engineering and regenerative medicine.