技术资料

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

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

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

STUDY HYPOTHESIS Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture,often accompanied by erosion of XCI-specific methylation,and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY Variable XCI patterns have been reported in female hPSC,not only between different hPSC lines,but also between sub-passages of the same cell line,however the reasons for this variability remain unknown. Moreover,while non-random XCI-linked DNA methylation patterns have been previously reported,their origin and extent have not been investigated. STUDY DESIGN,SAMPLES/MATERIALS,METHODS We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines,during long-term culture and after differentiation,by gene expression analysis,histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence,XIST expression by real-time PCR,and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR,and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE All hPSC lines showed XCI,but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation,it did not result in X chromosome reactivation. Moreover,lines without strong erosion of methylation frequently displayed non-random DNA methylation,which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore,we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally,differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS,REASONS FOR CAUTION All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC,more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen,grant 1502512N),Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel,on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest. View Publication

Fluorescence in situ hybridization (FISH) is superior to routine chromosome analysis (RCA) in detecting important prognostic genetic abnormalities in plasma cell dyscrasia (PCD); however,its sensitivity is hampered due to paucity of plasma cells (PC) in whole bone marrow (BM). Studies showed that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells (UPC),but purification techniques are limiting to only FISH when sample volumes are inadequate. Not performing RCA may compromise patient care since RCA is equally important for detecting non-PC related abnormalities when the diagnosis is undefined. To resolve this critical issue,we designed a study where an immuno-magnetic CD138 enriched positive selection was used for FISH while the negative fraction (NF) was used to retrieve other myeloid elements for RCA. Parallel FISH studies were performed using UPC and CD138 EPC,while karyotyping was achieved using whole BM and discarded myeloid elements from the NF. Results showed that the abnormality rate of EPC was doubled compared to UPC for FISH,and CA displayed 100% success rate using the NF. PCD related chromosome abnormalities were confined to whole BM while non-PCD related abnormalities were found in both whole BM and NF. Our results demonstrate the feasibility of using the NF for RCA. View Publication

Resveratrol (trans-3,5,4'-trihydroxystilbene) (RSV) is a natural polyphenol with protective effects over cardiac tissues and can affect cell survival and differentiation in cardiac stem cells transplantation. However,whether this agent can affect cardiomyocytes (CMs) differentiation of induced pluripotent stem cells (iPSCs) is not yet clear. This study explored whether RSV can affect CMs differentiation of human iPSCs. Under embryoid bodies (EBs) condition,the effect of RSV on the change of pluripotent markers,endoderm markers,mesoderm markers,and ectoderm markers was measured using qRT-PCR. Under CM differentiation culture,the effect of RSV on CM specific markers was also measured. The regulative role of RSV over canonical Wnt signal pathway and serum response factor- (SRF-) miR-1 axis and the functions of these two axes were further studied. Results showed that RSV had no effect on the self-renewal of human iPSCs but could promote mesoderm differentiation. Under CM differentiation culture,RSV could promote CM differentiation of human iPSCs through suppressing canonical Wnt signal pathway and enhancing SRF-miR-1 axis. View Publication

Upon infection,antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor,the core component of mammalian target of rapamycin complex 2 (mTORC2),regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover,mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation,repression of T-bet,enhanced mitochondrial spare respiratory capacity,and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore,mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions. View Publication

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening,disease modeling,and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture,such as a stirred bioreactor,are generally considered as promising approaches to produce the required cells. Recently,suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling,showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification,3-D neural tissue development,or potential preclinical studies or clinical applications in neurological diseases. View Publication

BACKGROUND Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus,tumor-initiating cell-like cells are generated. METHODS We employed laser-induced single-cell fusion technique to fuse the hepatocellular carcinoma cells and human embryonic stem cells (hESC). Real-time RT-PCR,flow cytometry and in vivo tumorigenicity assay were adopted to identify the gene expression difference. RESULTS We successfully produced a fused cell line that coalesces the gene expression information of hepatocellular carcinoma cells and stem cells. Experimental results showed that the fused cells expressed cancer and stemness markers as well as exhibited increased resistance to drug treatment and enhanced tumorigenesis. CONCLUSIONS Fusion with stem cells transforms liver cancer cells into tumor initiating-like cells. Results indicate that fusion between cancer cell and stem cell may generate tumor initiating-like cells. View Publication

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues,which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases,appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study,we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS),or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally,calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next,we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs,and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions. View Publication

BACKGROUND Rhodiola crenulata is a Tibetan mountainous plant,commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity,a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells,lacking the expression of estrogen receptor. Currently,it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE In this study,we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally,to assess stem-like properties in MCF7 cells,we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced $$-catenin levels via Western blotting and measured $$-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells,we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS We show an initial activation of ER transcriptional activity by dual reporter assay,qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However,upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes,ER$$ transcript levels were decreased and active $$-catenin levels were reduced in the cells treated for 2 weeks. Finally,we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION These data suggest that the R. crenulata extract contains components with estrogenic activity. However,R. crenulata treatment could still be protective in ER+ breast cancer cells,as longer treatment reduced the transcriptional activity of $$-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore,administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ER$$ target gene expression in vivo. View Publication

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication