技术资料

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

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The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

The multidrug transporter ABCG2 in cell membranes enables various stem cells and cancer cells to efflux chemicals,including the fluorescent dye Hoechst 33342. The Hoechst(-) cells can be sorted out as a side population with stem cell properties. Abcg2 expression in mouse embryonic stem cells (ESCs) reduces accumulation of DNA-damaging metabolites in the cells,which helps prevent cell differentiation. Surprisingly,we found that human ESCs do not express ABCG2 and cannot efflux Hoechst. In contrast,trophoblasts and neural epithelial cells derived from human ESCs are ABCG2(+) and Hoechst(-). Human ESCs ectopically expressing ABCG2 become Hoechst(-),more tolerant of toxicity of mitoxantrone,a substrate of ABCG2,and more capable of self-renewal in basic fibroblast growth factor (bFGF)-free condition than control cells. However,Hoechst(low) cells sorted as a small subpopulation from human ESCs express lower levels of pluripotency markers than the Hoechst(high) cells. Similar results were observed with human induced pluripotent stem cells. Conversely,mouse ESCs are Abcg2(+) and mouse trophoblasts,Abcg2(-). Thus,absence of ABCG2 is a novel feature of human pluripotent stem cells,which distinguishes them from many other stem cells including mouse ESCs,and may be a reason why they are sensitive to suboptimal culture conditions. View Publication

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications,including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs),a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC),and also the gold standard human embryonic stem cell,we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness,and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly,cells exhibited a noticeable difference in stiffness. From least to most stiff,the order of cell stiffness was as follows: hASC-iPSC,human embryonic stem cell,fibroblast-iPSC,fibroblasts,and,lastly,as the stiffest cell,hASC. In comparing hASC-iPSCs to their origin cell,the hASC,the reprogrammed cell is significantly less stiff,indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence,material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state. View Publication

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo,but after isolation can be expanded considerably,generating clinically useful numbers of cells. In this study,we demonstrate the use of a defined embryonic stem cell expansion medium,mTeSR (Stem Cell Technologies),for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates,demonstrate tri-lineage differentiation potential,and show similar surface marker profiles (CD29(+),CD44(+),CD49a(+),CD73(+),CD90(+),CD105(+),CD146(+),CD166(+),CD34(-),and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However,expression of early differentiation transcription factors runt-related transcription factor 2,sex-determining region Y box 9,and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated,whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS,adipogenic differentiation was significantly decreased in mTeSR-expanded cells,both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further,this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs,such as bone repair and cartilage generation. View Publication

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells,yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes,but how much epigenomes differ remains unclear. Here,we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts,we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks,which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally,we observe novel,context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment. textcopyright 2010 Elsevier Inc. View Publication

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types,and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. View Publication

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was,for the first time,to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined,and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC,Osx,and Runx2 genes,both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group,histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering,by promoting the formation of new cementum,alveolar bone,and normal periodontal ligament. View Publication

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function,little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages,to migrate toward stromal cell-derived factor 1α,and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However,the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast,adhesion to the extracellular matrix was inhibited by silencing of talin-1,but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity,independent of integrin functions. View Publication

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus,rs12740374,creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver,we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus,we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes. View Publication

Until recently,culturing human pluripotent stem cells was hampered by three prominent technical problems: a high degree of unwanted cellular stress when the cells are passaged,unacceptably low cloning efficiency and poor recovery of cryopreserved stocks. This review discusses recent developments that address these problems. A major focus of the review is the use of p160 Rho-associated coiled-coil kinase inhibitors for improving both the cloning efficiency and the recovery of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. An underlying theme of this review is that the three problems have a common cause: separation of human pluripotent stem cells from one another increases cellular stress,which greatly decreases their viability unless special steps are taken. View Publication