技术资料

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication

BACKGROUND: The radiation-induced bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However� View Publication

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

BACKGROUND Many groups have generated insulin-secreting cells from hESCs/iPSCs in multiple differentiation stages by mimicking the developmental processes. However,these cells do not always secrete glucose responsive insulin,one of the most important characteristics of pancreatic $$ cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic $$ cells. METHODS We established a 6-stage protocol for the differentiation process from hiPSCs to pancreatic $$ cells using defined culture media without feeders or serum. We examined the effect of CHIR99021,the selective inhibitor of GSK-3$$,in the presence of Activin,FGF2,and BMP4 during definitive endodermal induction by immunostaining for SOX17 and FOXA2. We also compared the insulin secretion at the last stage between monolayer culture and spheroid culture conditions. Cultured cells were transplanted under the kidney capsules of STZ-induced diabetic NOD-SCID mice,and blood glucose levels were measured. Immunohistochemical analysis was performed 4 weeks and 12 weeks after transplantation. RESULTS Addition of CHIR99021 in the presence of Activin,FGF2,and BMP4 for 2 days improved the viability of the endodermal cells,keeping the high positive rate of SOX17. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than monolayer culture did. After cell transplantation,diabetic mice showed lowered blood glucose levels,and we detected islet-like structures in vivo. CONCLUSION We generated functional pancreatic $$ cells from human iPS cells. Induction of definitive endoderm and spheroid formation might be key steps for producing them. View Publication

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

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7),the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions,and currently,there are no effective forms of therapy. Nevertheless,some advances in patient therapy are being made,and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized,gene-corrected,patient-specific cell transfer,we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly,human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis. View Publication

More than 10 years after their first isolation,human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable,and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever,biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper,we review the cutting edge of this exciting area of translational research. View Publication

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH),a DA neuron marker,and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth. View Publication

In guiding hES cell technology toward the clinic,one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So,we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture,including the developmental potential to differentiate into representative tissues of all three embryonic germ layers,unlimited and undifferentiated proliferative ability,and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF,insulin-like growth factor 2 (IGF-2),and transforming growth factor β (TGF-β),thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together,bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically. View Publication

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However,it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines,along with methylomes of ES cells,somatic cells,and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability,including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation,and differences in CG methylation and histone modifications. Lastly,differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency,providing an iPSC reprogramming signature that is maintained after differentiation. View Publication