技术资料

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication

gamma-Secretase is a membrane-associated protease with multiple intracellular targets,a number of which have been shown to influence embryonic development and embryonic stem (ES) cell differentiation. This paper describes the use of the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) to evaluate the role of gamma-secretase in the differentiation of pluripotent stem cells to the germ lineages. The addition of DAPT did not prevent the formation of primitive ectoderm-like cells from ES cells in culture. In contrast,the addition of DAPT during primitive ectoderm-like cell differentiation interfered with the ability of both serum and BMP4 to induce a primitive streak-like intermediate and resulted in the preferential formation of neurectoderm. Similarly,DAPT reduced the formation of primitive streak-like intermediates from differentiating human ES cells; the culture conditions used resulted in a population enriched in human surface ectoderm. These data suggest that gamma-secretase may form part of the general pathway by which mesoderm is specified within the primitive streak. The addition of an E-cadherin neutralizing antibody was able to partially reverse the effect of DAPT,suggesting that DAPT may be preventing the formation of primitive streak-like intermediates and promoting neurectoderm differentiation by stabilizing E-cadherin and preventing its proteolysis. View Publication

Recent studies support the notion that there is an intricate relationship between hematopoiesis and bone homeostasis in normal steady states. Using mice undergoing chronic inflammatory arthritis,we investigated the relationship between hematopoiesis and bone homeostasis in pathologic conditions. We demonstrate that mice undergoing chronic inflammatory arthritis displayed osteoporosis resulting from a severe defect in osteoblast function. Despite the defective osteoblast function,however,the hematopoietic stem cells from these mice exhibited normal properties in either long-term repopulation or cell cycling. Therefore,the bone-forming capacity of osteoblasts is distinct from their ability to maintain hematopoietic stem cells in chronic inflammatory conditions. View Publication

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Stem cells derived from pre-implantation human embryos or from somatic cells by reprogramming are pluripotent and self-renew indefinitely in culture. Pluripotent stem cells are unique in being able to differentiate to any cell type of the human body. Differentiation towards the cardiac lineage has attracted significant attention,initially with a strong focus on regenerative medicine. Although an important research area,the heart has proven challenging to repair by cardiomyocyte replacement. However,the ability to reprogramme adult cells to pluripotent stem cells and genetically manipulate stem cells presented opportunities to develop models of human disease. The availability of human cardiomyocytes from stem cell sources is expected to accelerate the discovery of cardiac drugs and safety pharmacology by offering more clinically relevant human culture models than presently available. Here we review the state-of-the-art using stem cell-derived human cardiomyocytes in drug discovery,drug safety pharmacology,and regenerative medicine. View Publication

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However,there was no systematic comparison of supportive effects of the feeder cells on hESC growth,nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study,we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF,and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs. View Publication

Since mouse embryonic stem (ES) cells was first derived in 1981,the ability of this unprecedented cell type to self-renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore,they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest,due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However,this is no longer a barrier,with the recent discovery of methods that can convert differentiated somatic cells into ES-like cells or induced pluripotent stem (iPS) cells,by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo-derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells,as well as the different determinants identified thus far to sustain their pluripotency. View Publication

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells,and Ras activity enhances the oncogenic ability of Bcr-Abl. However,the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction,resulting in blocking the MAP kinase pathway. In the present study,we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl(+) progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status,resulting in inhibited proliferation of CML cells. Moreover,RKIP up-regulated cell cycle regulator FoxM1 expression,resulting in G(1) arrest via p27(Kip1) and p21(Cip1) accumulation. In colony-forming unit granulocyte,erythroid,macrophage,megakaryocyte,colony-forming unit-granulocyte macrophage,and burst-forming unit erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions,and inhibited colony formation of Bcr-Abl(+) progenitor cells,whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl(+) progenitor cells. Thus,Bcr-Abl represses the expression of RKIP,continuously activates pERK1/2,and suppresses FoxM1 expression,resulting in proliferation of CML cells. View Publication