技术资料

Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation,our protocol differs in the combination of commercial availability of all reagents,technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d. View Publication

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes,and forced expression of OCT4,KLF4,and KLF2 allows maintenance of human cells in a naïve state [Hanna J,et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid,followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics,antibody labeling profile,gene expression,X-inactivation profile,mitochondrial morphology,microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive,but attainable,process,leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible. View Publication

Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells,which will be useful for experimental application. View Publication

Embryonic and induced pluripotent stem cells (IPSCs) derived from mammalian species are valuable tools for modeling human disease,including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. Here we used transgenic common marmoset (Callithrix jacchus) and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP) reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures,we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications. View Publication

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation,and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI,and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I),we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI,we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation,and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together,our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation,and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. View Publication

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts,and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer,retaining a pre-inactivation X chromosome state,and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF,naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells,they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression,pronounced tendency for X chromosome inactivation in most female human ES cells,increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells,from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells,and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively,our findings establish new avenues for regenerative medicine,patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. View Publication

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition,patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless,directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis,which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here,we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes,enabling researchers to use these cells for disease modeling as well as therapeutic purposes. View Publication

Recently,induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer,both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore,the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front,although donor cell types for generating iPSCs are wide-ranging,T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells,which can be applied to novel immunotherapies. In the present study,we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining,had a normal karyotype,and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined,animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application. View Publication

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA,PARK2,LRRK2,and GBA mutations,and one age-matched control. After validation of pluripotency,long-term genome stability,and integration-free reprogramming,eight of these lines (one of each SNCA,LRRK2 and GBA,four PARK2 lines,and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line,nor amongst the patient lines,although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast,iPSC,NSC,and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation,no mutation-specific clustering or difference was observed,though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line,and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease,and highlights the need to use more focused strategies for large-scale data analysis. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid,guggulsterone,was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients. View Publication

Despite major progress in the management of human liver disease,the only cure for a critically failing organ is liver transplantation. While a highly successful approach,the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore,the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments,there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process,offering new treatment or preventative strategies. With both approaches in mind,we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly,our procedure is highly efficient (∼ 90%) and delivers active,drug-inducible,and predictive human hepatocyte populations. View Publication