技术资料

MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However,the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here,by using system biology approaches,we show for the first time,that miRNAome undergoes global alterations in hESC (H1 and H9 lines) after IR. Interrogation of expression levels of 1,090 miRNA species in irradiated hESC showed statistically significant changes in 54 genes following 1 Gy of X-ray exposures; global miRNAome alterations were found to be highly temporally and cell line--dependent in hESC. Time-course studies showed that the 16 hr miRNAome radiation response of hESC is much more robust compared to 2 hr-response signature (only eight genes),and may be involved in regulating the cell cycle. Quantitative real-time PCR performed on some miRNA species confirms the robustness of our miRNA microarray platform. Positive regulation of differentiation-,cell cycle-,ion transport- and endomembrane system-related processes were predicted to be negatively affected by miRNAome changes in irradiated hESC. Our findings reveal a fundamental role of miRNAome in modulating the radiation response,and identify novel molecular targets of radiation in hESC. View Publication

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood,and biomarkers to identify those at risk are lacking. Here,we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure. METHODS: WA01 and WA09 hES cells were differentiated into embryoid bodies,neural progenitors,or neurons. Cells along this progression were cultured for 4 days with 0,0.1,or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases,and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS. RESULTS: EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features),neural progenitors (76 features),and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine,5'-methylthioadenosine,and the tryptophan metabolites,L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases. CONCLUSIONS: EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies,neural progenitors,and neurons. Several of these metabolites are normally present in human serum,suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development. View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product,the amyloid-beta (Abeta) protein,is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease,Down syndrome,and head injury. We recently reported that this protein,normally associated with neurodegenerative conditions,is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta,which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression,an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha,which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis. View Publication

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BACKGROUND The incidence of neurological complications and fatalities associated with Hand,Foot & Mouth disease has increased over recent years,due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71,accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite,before progressing to expensive and time-consuming live animal studies and clinical trials. METHODS This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro,in comparison with RD and SH-SY5Y cell lines. RESULTS Upon assessment of post-infection survivability and EV71 production by the various types,it was observed that NSC were significantly more susceptible to EV71 infection compared to MN,RD (rhabdomyosarcoma) and SH-SY5Y cells,which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. CONCLUSIONS Hence,this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics. View Publication

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

Since the discovery of induced pluripotent stem cells (iPSCs),numerous approaches have been explored to improve the original protocol,which is based on a two-dimensional (2D) cell-culture system. Surprisingly,nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here,we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness,degradability and biochemical composition,we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity. View Publication

Human pluripotent stem cell (hPSC) density is an important factor in self-renewal and differentiation fates; however,the mechanisms through which hPSCs sense cell density and process this information in making cell fate decisions remain to be fully understood. One particular pathway that may prove important in density-dependent signaling in hPSCs is the Hippo pathway,which is regulated by cell-cell contact and mechanosensing through the cytoskeleton and has been linked to the maintenance of stem cell pluripotency. To probe regulation of Hippo pathway activity in hPSCs,we assessed whether Hippo pathway transcriptional activator YAP was differentially modulated by cell density. At higher cell densities,YAP phosphorylation and localization to the cytoplasm increased,which led to decreased YAP-mediated transcriptional activity. Furthermore,total YAP protein levels diminished at high cell density due to the phosphorylation-targeted degradation of YAP. Inducible shRNA knockdown of YAP reduced expression of YAP target genes and pluripotency genes. Finally,the density-dependent increase of neuroepithelial cell differentiation was mitigated by shRNA knockdown of YAP. Our results suggest a pivotal role of YAP in cell density-mediated fate decisions in hPSCs. View Publication

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here,for the first time,human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells,exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture,the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs,suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However,continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells,linking embryonic genotoxic exposure to genomic instability. View Publication

BACKGROUND: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity,molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock,a major effector kinase downstream of Rho,played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore,a novel culture system combining a single synthetic matrix,defined medium,and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices,tight cell contacts,or fibroblastic niche-forming cells as determined by teratoma formation assay.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells,and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells. View Publication

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However,currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability,painful and invasive cell-harvesting procedures,and tumorigenesis. Previously,we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan,fibromodulin (FMOD),circumventing gene transduction. In this study,we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect,contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence,engraftment,and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together,we have provided an extended potency,safety,and molecular profile of FReP cell-based bone regeneration. Therefore,FReP cells present a high potential for cellular and gene therapy products for bone regeneration. View Publication

Type II diabetes mellitus (T2D) is a chronic metabolic disorder that results from defects in both insulin secretion and insulin action. The deficit and dysfunction of insulin secreting $\$-cell are signature symptom for T2D. Additionally,in pancreatic $\$-cell,a small group of genes which are abundantly expressed in most other tissues are highly selectively repressed. Lactate dehydrogenase A (LDHA) is one of such genes. Upregulation of LDHA is found in both human T2D and rodent T2D models. In this study,we identified a LDHA-suppressing microRNA (hsa-miR-590-3p) and used it together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) transplantation into a high-fat diet induced T2D mouse model. The procedure significantly improved glucose metabolism and other symptoms of T2D. Our findings support the potential T2D treatment using the combination of microRNA and hESC-differentiated PE cells. View Publication