技术资料

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

In this study,we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy,qPCR and flow cytometry,we classified the treatment outcome as inducing pluripotency (hESC,flurbiprofen and gatifloxacin),mesendoderm (sinomenine),differentiation (cyamarin,digoxin,digitoxin,selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions,the above classification was reassorted. Hyperacetylation at H3K4,9,14,18,56 and 122 as well as H4K5,8,12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9,K20,K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to differentiation initiators"� View Publication

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG),affecting many cell types including those of the pancreas. Indeed,XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin,insulin,and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development,we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected),glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult $$-cells. Differentiated ARX knockout cells upregulated PAX4,NKX2.2,ISL1,HHEX,PCSK1,PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide,somatostatin,glucagon and insulin positive cells from hESCs. View Publication

The tentative clinical application of human pluripotent stem cells (hPSCs),such as human embryonic stem cells and human induced pluripotent stem cells,is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore,we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture,whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture. View Publication

AIM To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology,expression of corneal endothelial markers,and microarray analysis of gene expression. RESULTS hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells,expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPase$\$1 (ATPA1) on the apical surface in monolayer culture,and produced the key proteins of Descemet's membrane,Collagen VIII$\$1 and VIII$\$2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. CONCLUSION hESC-CECs are morphologically similar,express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. View Publication

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However,how it mediates Ca2+ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here,we aimed to determine the role of P2Rs in mediating Ca2+ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations,respectively. Confocal imaging revealed that Ca2+ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently,the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca2+ transients in hESCs but only partially inhibited those in CVPCs. Moreover,the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca2+ signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca2+ transients only in hESCs but not in CVPCs. Furthermore,IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast,both IP3R2 and IP3R3 contributed to UTP-induced Ca2+ responses while ATP-induced Ca2+ responses were more dependent on IP3R2 in the CVPCs. In conclusion,a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca2+ mobilization between these cells. View Publication

Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example,human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes,respectively. However,the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation,FGF/Wnt-induced posterior endoderm pattering,hindgut specification and morphogenesis,and a pro-intestinal culture system to promote intestinal growth,morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized,columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes,as well as goblet,Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development,we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3,a pro-endocrine transcription factor that is mutated in enteric anendocrinosis,is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease. View Publication

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However,this is a slow and inefficient process,depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover,once cell reprogramming is accomplished,these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However,no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus,which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes,deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore,interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus,this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. View Publication

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here,we show dynamic recruitment of GATA-1,TFIIB,Mediator,and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice,we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development,highlighted by the complete absence of β-globin gene expression. Thus,Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation. View Publication

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD),including tuberous sclerosis,caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here,we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism,suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis. View Publication

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However,functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC,a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover,strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα),were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors,and may contribute to the block in definitive hematopoiesis from hESC. View Publication