技术资料

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology� View Publication

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton,cornea,peripheral nervous system,and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),further modifications are required to improve the robustness,efficacy,and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions,the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons,glia,melanocytes,and corneal endothelial cells. In addition,cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine. 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

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

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

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery,mutagenesis,and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives,pZR-1 and pZR-2,that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene,either beta-lactamase or enhanced green fluorescent protein (EGFP),that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream,cell-specific promoter during cell culture and cell differentiation,which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment,as well as for the discovery of new genes. View Publication