Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming.

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.

Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells.

Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed "cis-silenced" (or "occluded") genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.

Directed differentiation of human embryonic stem cells to corneal endothelial cell-like cells: A transcriptomic analysis.

Corneal endothelial cells (CECs) are a monolayer of cells covering the inner-side of cornea, playing a pivotal role in keeping the cornea transparent. Because adult CECs have no proliferative capacity, the loss of CECs during aging or under pathological conditions would lead to corneal edema, eventually leading to the blindness. Clinically, donated CECs have been successfully transplanted to treat the diseases of CEC deficiency; however, the source of CEC donation is very limited. As an alternative cell source for CEC transplantation, CEC-like cells can be obtained via in vitro differentiation of human pluripotent stem cells. In this study, we introduced a modified two-stage differentiation method to convert H9 human embryonic stem cells (hESCs) to neural crest cells (NCCs), then further into CEC-like cells. The CEC-like cells treated with bovine CEC conditional medium morphologically best resembled primary CECs among all the culture conditions. By whole transcriptome analysis, we found that the typical markers of CECs, such as Na+-K+-ATPase, AQP1, Col8a and ZO-1, are highly expressed in hESC-derived CEC-like cells. By comparing RNA transcriptome of hESC-derived CEC-like cells with human primary fetal and adult CECs, we further identified shared molecular markers such as TRIT1, HSPB11, CRY1 that can be used to quality control CEC derivatives from hESCs. Our study paves the way for the quality-control and future application of hESC-derived CECs in the treatment of CEC deficiency disorders.

AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure.

Leydig cell failure (LCF) caused by gene mutation results in testosterone deficiency and infertility. Serum testosterone levels can be recovered via testosterone replacement; however, established therapies have shown limited success in restoring fertility. Here, we use a luteinizing hormone/choriogonadotrophin receptor (Lhcgr)-deficient mouse model of LCF to investigate the feasibility of gene therapy for restoring testosterone production and fertility. We screen several adeno-associated virus (AAV) serotypes and identify AAV8 as an efficient vector to drive exogenous Lhcgr expression in progenitor Leydig cells through interstitial injection. We observe considerable testosterone recovery and Leydig cell maturation after AAV8-Lhcgr treatment in pubertal Lhcgr-/- mice. Of note, this gene therapy partially recovers sexual development, substantially restores spermatogenesis, and effectively produces fertile offspring. Furthermore, these favorable effects can be reproduced in adult Lhcgr-/- mice. Our proof-of-concept experiments in the mouse model demonstrate that AAV-mediated gene therapy may represent a promising therapeutic approach for patients with LCF.

Nestin is required for the proper self-renewal of neural stem cells.

The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self-renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestin's ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self-renewal of NSCs, and that this function is surprisingly uncoupled from nestin's structural involvement in the cytoskeleton.

Autologous transplantation of thecal stem cells restores ovarian function in nonhuman primates.

Premature ovarian insufficiency (POI) is defined as the loss of ovarian activity under the age of 40. Theca cells (TCs) play a vital role during folliculogenesis and TCs dysfunction participate in the pathogenesis of POI. Therefore, transplantation of thecal stem cells (TSCs), which are capable of self-renewal and differentiation into mature TCs, may provide a new strategy for treating POI. To investigate the feasibility, safety, and efficacy of TSCs transplantation in clinically relevant non-human primate (NHP) models, we isolate TSCs from cynomolgus monkeys, and these cells are confirmed to expand continuously and show potential to differentiate into mature TCs. In addition, engraftment of autologous TSCs into POI monkeys significantly improves hormone levels, rescues the follicle development, promotes the quality of oocytes and boosts oocyte maturation/fertilization rate. Taken together, these results for the first time suggest that autologous TSCs can ameliorate POI symptoms in primate models and shed new light on developing stem cell therapy for POI.

Generation of functional hepatocytes from mouse induced pluripotent stem cells.

Induced pluripotent stem cells are derived from somatic cells by forced expression of several transcriptional factors. Induced pluripotent stem cells resemble embryonic stem cells in many aspects, such as the expression of certain stem cell markers, chromatin methylation patterns, embryoid body formation and teratoma formation. Therefore, induced pluripotent stem cells provide a powerful tool for study of developmental biology and unlimited resources for transplantation therapy. Here we reported the successful induction of mouse induced pluripotent stem cells and a simple and efficient process for generation of functional hepatocytes from mouse induced pluripotent stem cells by sequential addition of inducing factors. These induced pluripotent stem cell-derived hepatocytes, just as mouse embryonic stem cell-derived hepatocytes, expressed hepatic lineage markers including CK7, CK8, CK18, CK19, alpha-fetoprotein, albumin, Cyp7a1, and exhibited functional hepatic characteristics, including glycogen storage, indocyanine green (ICG) uptake and release, low-density lipoprotein (LDL) uptake and urea secretion. Although we observed some variations in the efficiency of hepatic differentiation between induced pluripotent stem cells and common mouse embryonic stem cell lines, our results indicate that mouse induced pluripotent stem cells can efficiently differentiate into functional hepatocytes in vitro, which may be helpful for the study of liver development and regenerative medicine.

Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro.

BACKGROUND: Embryonic stem (ES) cells have attracted significant attention from researchers around the world because of their ability to undergo indefinite self-renewal and produce derivatives from the three cell lineages, which has enormous value in research and clinical applications. Until now, many ES cell lines of different mammals have been established and studied. In addition, recently, AS-ES1 cells derived from Apodemus sylvaticus were established and identified by our laboratory as a new mammalian ES cell line. Hence further research, in the application of AS-ES1 cells, is warranted. RESULTS: Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB) formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells. CONCLUSIONS: The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.

Derivation, characterization and gene modification of cynomolgus monkey mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have received considerable attention in recent years. Particularly exciting is the prospect that MSCs could be differentiated into specialized cells of interest, which could then be used for cell therapy and tissue engineering. MSCs derived from nonhuman primates could be a powerful tool for investigating the differentiation potential in vitro and in vivo for preclinical research. The purpose of this study was to isolate cynomolgus mesenchymal stem cells (cMSCs) from adult bone marrow and characterize their growth properties and multipotency. Mononuclear cells were isolated from cynomolgus monkey bone marrow by density-gradient centrifugation, and adherent fibroblast-like cells grew well in the complete growth medium with 10 microM Tenofovir. cMSCs expressed mesenchymal markers, such as CD29, CD105, CD166 and were negative for hematopoietic markers such as CD34, CD45. Furthermore, the cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages under certain conditions, maintaining normal karyotype throughout extended culture. We also compared different methods (lipofection, nucleofection and lentivirus) for genetic modification of cMSCs and found lentivirus proved to be the most effective method with transduction efficiency of up to 44.6% and lowest level of cell death. The cells after transduction stably expressed green fluorescence protein (GFP) and maintained the abilities to differentiate down osteogenic and adipogenic lineages. In conclusion, these data showed that cMSCs isolated from cynomolgus bone marrow shared similar characteristics with human MSCs and might provide an attractive cell type for cell-based therapy in higher-order mammalian species disorder models.

The radial glia antibody RC2 recognizes a protein encoded by Nestin.

The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin-/- cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).

Enhanced generation of human induced pluripotent stem cells by ectopic expression of Connexin 45.

Somatic cells can be successfully reprogrammed into pluripotent stem cells by the ectopic expression of defined transcriptional factors. However, improved efficiency and better understanding the molecular mechanism underlying reprogramming are still required. In the present study, a scrape loading/dye transfer assay showed that human induced pluripotent stem cells (hiPSCs) contained functional gap junctions partially contributed by Connexin 45 (CX45). We then found CX45 was expressed in human embryonic stem cells (hESCs) and human dermal fibroblasts (hDFs) derived hiPSCs. Then we showed that CX45 was dramatically upregulated during the reprogramming process. Most importantly, the ectopic expression of CX45 significantly enhanced the reprogramming efficiency together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM), whereas knockdown of endogenous CX45 expression significantly blocked cellular reprogramming and reduced the efficiency. Our further study demonstrated that CX45 overexpression or knockdown modulated the cell proliferation rate which was associated with the reprogramming efficiency. In conclusion, our data highlighted the critical role of CX45 in reprogramming and may increase the cell division rate and result in an accelerated kinetics of iPSCs production.

Neural differentiation of embryonic stem cells induced by conditioned medium from neural stem cell.

Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.

TALEN-based generation of a cynomolgus monkey disease model for human microcephaly.

Gene editing in non-human primates may lead to valuable models for exploring the etiologies and therapeutic strategies of genetically based neurological disorders in humans. However, a monkey model of neurological disorders that closely mimics pathological and behavioral deficits in humans has not yet been successfully generated. Microcephalin 1 (MCPH1) is implicated in the evolution of the human brain, and MCPH1 mutation causes microcephaly accompanied by mental retardation. Here we generated a cynomolgus monkey (Macaca fascicularis) carrying biallelic MCPH1 mutations using transcription activator-like effector nucleases. The monkey recapitulated most of the important clinical features observed in patients, including marked reductions in head circumference, premature chromosome condensation (PCC), hypoplasia of the corpus callosum and upper limb spasticity. Moreover, overexpression of MCPH1 in mutated dermal fibroblasts rescued the PCC syndrome. This monkey model may help us elucidate the role of MCPH1 in the pathogenesis of human microcephaly and better understand the function of this protein in the evolution of primate brain size.

Evidence for a critical role of gene occlusion in cell fate restriction.

The progressive restriction of cell fate during lineage differentiation is a poorly understood phenomenon despite its ubiquity in multicellular organisms. We recently used a cell fusion assay to define a mode of epigenetic silencing that we termed "occlusion", wherein affected genes are silenced by cis-acting chromatin mechanisms irrespective of whether trans-acting transcriptional activators are present. We hypothesized that occlusion of lineage-inappropriate genes could contribute to cell fate restriction. Here, we test this hypothesis by introducing bacterial artificial chromosomes (BACs), which are devoid of chromatin modifications necessary for occlusion, into mouse fibroblasts. We found that BAC transgenes corresponding to occluded endogenous genes are expressed in most cases, whereas BAC transgenes corresponding to silent but non-occluded endogenous genes are not expressed. This indicates that the cellular milieu in trans supports the expression of most occluded genes in fibroblasts, and that the silent state of these genes is solely the consequence of occlusion in cis. For the BAC corresponding to the occluded myogenic master regulator Myf5, expression of the Myf5 transgene on the BAC triggered fibroblasts to acquire a muscle-like phenotype. These results provide compelling evidence for a critical role of gene occlusion in cell fate restriction.

A stem cell-based tool for small molecule screening in adipogenesis.

Techniques for small molecule screening are widely used in biological mechanism study and drug discovery. Here, we reported a novel adipocyte differentiation assay for small molecule selection, based on human mesenchymal stem cells (hMSCs) transduced with fluorescence reporter gene driven by adipogenic specific promoter--adipocyte Protein 2 (aP2; also namely Fatty Acid Binding Protein 4, FABP4). During normal adipogenic induction as well as adipogenic inhibition by Ly294002, we confirmed that the intensity of green fluorescence protein corresponded well to the expression level of aP2 gene. Furthermore, this variation of green fluorescence protein intensity can be read simply through fluorescence spectrophotometer. By testing another two small molecules in adipogenesis--Troglitazone and CHIR99021, we proved that this is a simple and sensitive method, which could be applied in adipocyte biology, drug discovery and toxicological study in the future.

Effects of thymic polypeptides on the thymopoiesis of mouse embryonic stem cells.

The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.

Extensive contribution of embryonic stem cells to the development of an evolutionarily divergent host.

The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.

Proteomic identification of differently expressed proteins responsible for osteoblast differentiation from human mesenchymal stem cells.

Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.