技术资料

(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells,FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM,noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme. View Publication

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic $\$-cells. In other systems,Musashi genes regulate cell fate via Notch signaling,which we recently showed regulates $\$-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms,as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of $\$-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1,while it decreased Ins1 and Ins2 expression,revealing a molecular link between ER stress and $\$-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1,stimulate proliferation,inhibit apoptosis and reduce insulin expression,whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together,these results demonstrate overlapping,but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and $\$-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory $\$-cell proliferation and the loss of $\$-cell gene expression seen in specific phases of the progression to type 2 diabetes. View Publication

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However,natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities,and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (˜50%) to hPSCs,which are importantly accompanied by a considerable increase in gene-targeting frequencies,up to 0.12%. While this level is likely sufficient for numerous applications,we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (textgreater1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus,this study demonstrates that under appropriate selective pressures,AAV vectors can be created to mediate efficient gene targeting in hPSCs,alone or in the presence of ZFN- mediated double-stranded DNA breaks. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper,we examined the dynamic and static micromechanical properties of hESC and hESC-CM,by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary,our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases. View Publication

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process,but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b,two known vitamin-C-dependent H3K36 demethylases,as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore,we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367,an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming. View Publication

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models,leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973,a novel selective MEK inhibitor,and GDC-0941,a class I PI3K inhibitor,are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here,we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice,we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis,including Bcl-2 family proapoptotic regulators. Therefore,these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing. View Publication

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application,but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin,dependent on PSC-CM maturity,and retained while PSC-CMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the non-invasive purification of PSC-CMs. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. View Publication