技术资料

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds,an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation,areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast,each with the potential to transport a wide range of solutes and synthesize placental hormones,their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. View Publication

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure,self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells,but its role in hESCs remains unclear. Here,we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly,knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly,FOXM1 depletion sensitized hESCs to oxidative stress. Moreover,genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1,which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together,our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs. View Publication

Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase,MLL2 (KMT2B),during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here,we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically,by utilizing dimeric CRISPR RNA-guided nucleases,RFNs (commercially known as the NextGEN™ CRISPR),in combination with an excision-only piggyBac™ transposase,we demonstrate how to generate a point mutation of threonine-542,a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection,and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations� View Publication

While in vitro liver tissue engineering has been increasingly studied during the last several years,presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver,but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly,generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions,and has been inefficient so far. Towards generating a fully functional liver containing biliary system,we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver,EpCAM,is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can,not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes),in a 2D differentiation condition,but also form functional ductal structures in a 3D condition. Importantly,this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition,we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues,which may facilitate engineering of complete and functional liver tissue in the future. View Publication

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation,reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study,we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter,tie-1. Using EGFP as a reporter gene,we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently,tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected,puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers,including CD31,CD34,VEGFR-1,VEGFR-2,Tie-1,VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1,two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally,we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together,these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies. View Publication

Human stem cells derived from human fertilized oocytes,fetal primordial germ cells,umbilical cord blood,and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However,the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach,employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D),we established stem cell lines homozygous for H-2-B and H-2-D,respectively. The undifferentiated cells retained a normal karyotype,expressed stage-specific embryonic antigen-1 and Oct4,and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition,these cells demonstrated the potential for in vitro differentiation into endoderm,neuronal,and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes,and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation� View Publication

Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However,the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells,SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study,we evaluated whether SS treatment could epigenetically modify histones and influence cell differentiation in mouse embryonic stem (ES) cells. Cells were exposed to a laminar SS of 10 dyne per cm2/s(-1),or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position 14 (K14),as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79),and cooperated with TSA,inducing acetylation of histone H4 and phosphoacetylation of S10 and K14 of histone H3. In addition,ES cells exposed to SS strongly activated transcription from the vascular endothelial growth factor (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers,including smooth muscle actin,smooth muscle protein 22-alpha,platelet-endothelial cell adhesion molecule-1,VEGF receptor 2,myocyte enhancer factor-2C (MEF2C),and alpha-sarcomeric actin. In this condition,transcription factors MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors. View Publication

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined,undifferentiated ESC in culture. In each dataset,we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets,despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest,we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728at,8430410A17Rik,Klf2,Nr0b1,Sox2,Tcl1,and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis,this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types. View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication

Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class I(A) PI3K isoforms has implicated PI3K-mediated signalling in development of the early embryo and lymphohaemopoietic system. We have used embryonic stem (ES) cells as an in vitro model to study the involvement of PI3K-dependent signalling during early development and haemopoiesis. Both pharmacological inhibition and genetic manipulation of PI3K-dependent signalling demonstrate that PI3K-mediated signals,most likely via 3-phosphoinositide-dependent protein kinase 1 (PDK1),are required for proliferation of cells within developing embryoid bodies (EBs). Surprisingly,the haemopoietic potential of EB-derived cells was not blocked upon PI3K inhibition but rather enhanced,correlating with modest increases in expression of haemopoietic marker genes. By contrast,PDK1-deficient EB-derived progeny failed to generate terminally differentiated haemopoietic lineages. This deficiency appeared to be due to a requirement for PI3K signalling during the proliferative phase of blast-colony-forming cell (BL-CFC) expansion,rather than as a result of effects on differentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation of erythroid and myeloid progenitors and their differentiation into mature haemopoietic colony types. These data demonstrate that PI3K-dependent signals play important roles at different stages of haemopoietic development. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

The enthusiasm surrounding the clinical potential of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is tempered by the fact that key issues regarding their safety,efficacy,and long-term benefits have thus far been suboptimal. Small molecules can potentially relieve these problems at major junctions of stem cell biology and regenerative therapy. In this review we will introduce recent advances in these important areas and the first generation of small molecules used in the regenerative context. Current chemical biology studies will provide the archetype for future interdisciplinary collaborations and improve clinical benefits of cell-based therapies. View Publication