技术资料

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation,purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages. View Publication

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication

Linker histones are essential components of chromatin,but the distributions and functions of many during cellular differentiation are not well understood. Here,we show that H1.5 binds to genic and intergenic regions,forming blocks of enrichment,in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells,H1.5,but not H1.3,binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly,37% of H1.5 target genes belong to gene family clusters,groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding,H3K9me2 enrichment,and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2,increased chromatin accessibility,deregulation of gene expression,and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells. View Publication

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms,yet their clinical translation has been compromised by their biosafety concern. Here,we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/ progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immu-nodeficient mice. Moreover,removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplanta-tion,ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable,depending on the oncogenic load,with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus,transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:694–702 SIGNIFICANCE Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products,especially when reprogrammed with integrating vectors. Two major under-lying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzy-matic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in test-ing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature b cell phenotype would lead to safe islet replacement therapy for diabetes. View Publication

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells,only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study,we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1,a critical transcription factor for pancreatic development,leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore,in the presence of biochemical factors,200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin,glucagon,or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ,suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. View Publication

The human naive pluripotent stem cell (PSC) state,corresponding to a pre-implantation stage of development,has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate,a normal karyotype,the ability to form teratomas,transcriptional similarities to human pre-implantation embryos,reduced heterochromatin levels,and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP-/- cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state,with implications for early human embryology. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here,we report the use of chitin-alginate 3D microfibrous scaffolds,previously developed for hPSC propagation,to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid,the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons,as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore,flow cytometry confirmed that textgreater90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines,a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons,without tumorigenesis,suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications. View Publication

The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work,we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB) derived from embryonic and induced pluripotent stem (ES and iPS) cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed,indicating that neural differentiation and elemental polarization are strongly correlated. View Publication

BACKGROUND Strategies on the basis of doxycycline-inducible lentiviruses in mouse cells allowed the examination of mechanisms governing somatic cell reprogramming. RESULTS Using a doxycycline-inducible human reprogramming system,we identified unreported miRs enhancing reprogramming efficiency. CONCLUSION We generated a drug-inducible human reprogramming reporter system as an invaluable tool for genetic or chemical screenings. SIGNIFICANCE These cellular systems provide a tool to enable the advancement of reprogramming technologies in human cells. Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years,reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene,driven by the reactivation of endogenous stem cell specific promoters,was used as a reprogramming reporter signal. However,similar reporter systems in human cells have not been generated. Here,we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system,we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency. View Publication

HIV-1 Tat-interacting protein of 110 kDa [Tip110; p110(nrb)/SART3/p110] is an RNA binding nuclear protein implicated in regulation of HIV-1 gene and host gene transcription,pre-mRNA splicing,and cancer immunology. Recently,we demonstrated a role for Tip110 in regulation of hematopoiesis. Here,we show that TIP110 is also expressed in human embryonic stem cells (hESCs) and expression was decreased with differentiation of these ESCs. TIP110 was found,through up- and down-modulation of expression of Tip110,to be important in maintaining pluripotent factor (NANOG,OCT4,and SOX2) expression in and pluripotency of hESCs,although the mechanisms involved and whether the Tip110 effects are direct remain to be determined. View Publication

Embryonic stem cells can replicate continuously in the absence of senescence and,therefore,are immortal in culture. Although genome stability is essential for the survival of stem cells,proteome stability may have an equally important role in stem-cell identity and function. Furthermore,with the asymmetric divisions invoked by stem cells,the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore,a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4,an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates,regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity. View Publication