技术资料

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations,gene expression profiles and response to treatment: WNT,Sonic Hedgehog (SHH),Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example,expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however,its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs),but not their normal counterparts (hENs),resemble Groups 3 and 4 MB in vitro and in vivo. Here,we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs,respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth,self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes,such as SOX2,and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast,OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. View Publication

The mechanisms underlying human germ cell development are largely unknown,partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here,we studied NANOS3 and DAZL,which have critical roles in germ cell development in several species,via their over expression in human embryonic stem cells using global transcriptional analysis,in vitro germ cell differentiation,and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition,we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL,our results suggest a post-transcriptional regulation mechanism in hES cells. In addition,we found that DAZL suppressed the translation of OCT4,and affected the transcription of several genes associated with germ cells,cell cycle arrest,and cell migration. Furthermore,DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. View Publication

OBJECTIVE Gastric cancer (GC) is one of the most common human cancers. A useful method of gastric cancer stem cell (CSC) characterization is spheroid colony formation. Previously,we reported that KIF11 expression is textgreater2-fold in spheroid-body-forming GC cells compared with parental cells. Here,we analyzed the expression and distribution of KIF11 in human GC by immunohistochemistry. METHODS Expression of KIF11 in 165 GC cases was determined using immunohistochemistry. For mucin phenotypic expression analysis of GC,immunostaining of MUC5AC,MUC6,MUC2 and CD10 was evaluated. RNA interference was used to inhibit KIF11 expression in GC cell lines. RESULTS In total,119 of 165 GC cases (72%) were positive for KIF11. Expression of KIF11 was not associated with any clinicopathologic characteristics; however,it was observed frequently in GC exhibiting an intestinal phenotype. Both the number and size of spheres formed by MKN-74 cells were significantly reduced following transfection of KIF11-targeting siRNA compared with negative-control siRNA. Furthermore,levels of phosphorylated Erk1/2 were lower in KIF11 siRNA-transfected cells than with negative-control siRNA-transfected cells. CONCLUSION These results indicate that KIF11 is involved in intestinal mucin phenotype GC. View Publication

Human embryonic stem cells (hESCs) are pluripotent and capable of generating new $\$-cells,but current in vitro differentiation protocols generally fail to produce mature,glucose-responsive,unihormonal $\$-cells. Instead,these methods tend to produce immature polyhormonal endocrine cells which mature in vivo into glucagon-positive $\$-cells. PAX4 is an established transcription factor in $\$-cell development and function,and is capable of converting glucagon-positive cells to insulin-positive cells in mice. Work in human and mouse ESCs has shown that constitutive PAX4 expression promotes the development of insulin-positive cells,but whether acute PAX4 expression is sufficient to guide specific endocrine cell fates has not been addressed in hESCs. In this study,we applied recombinant adenovirus to ectopically express human PAX4 in hESC-derived pancreatic progenitors,with the aim of influencing the endocrine developmental cascade away from polyhormonal cells toward unihormonal insulin-positive cells. Gene delivery to pancreatic progenitors was efficient and dose-dependent. By the end of in vitro differentiation,PAX4 reduced ARX expression,but only the high dose tested significantly reduced glucagon release. Single cell analysis revealed that while PAX4 did not alter the proportion of endocrine cells,it did reduce the number of glucagon-positive cells and increased the number of unihormonal insulin-positive cells. These data suggest that acute PAX4 overexpression can reduce expression of ARX and glucagon resulting in improved numbers of unihormonal insulin-positive cells. View Publication

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD),a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production,reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs,with subsequent selection and full characterization to ensure no off-target changes,holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs." View Publication

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet,cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation,viability and differentiation potential. Here,a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation,mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover,the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics. View Publication

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here,we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells,p53 in hESCs is maintained at low levels in the nucleus,albeit in a deacetylated,inactive state. In response to retinoic acid,CBP/p300 acetylates p53 at lysine 373,which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel,p53 activates expression of miR-34a and miR-145,which in turn repress stem cell factors OCT4,KLF4,LIN28A,and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation,whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs,independently of retinoic acid. Ectopic expression of p53R175H,a mutated form of p53 that does not bind DNA or regulate transcription,failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. View Publication

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here,we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D,etoposide,or tunicamycin,with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific,retinoic-acid-differentiated hPSCs. Also,Bax activation and pro-apoptotic mitochondrial intermembrane space protein release,which are required to initiate the mitochondria-mediated apoptosis pathway,are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly,Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally,P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether,we provide an underlying,interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development. View Publication

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects,we first established a liver organoid using human induced pluripotent stem cells (iPSCs),mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids,resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions,we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form,their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs,resulting in the expression of bile salt export pump. In conclusion,the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids,but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes. View Publication

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE,which encodes neutrophil elastase (NE). However,a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end,we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs),and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest,and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly,high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBP$$-dependent emergency granulopoiesis. In contrast,sivelestat,an NE-specific small-molecule inhibitor,corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA,but not CEBPB; and promoting promyelocyte survival and differentiation. Together,these data suggest that SCN disease pathogenesis includes NE mislocalization,which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization. View Publication

Skeletal dysplasias (SDs) are caused by abnormal chondrogenesis during cartilage growth plate differentiation. To study early stages of aberrant cartilage formation in vitro,we generated the first induced pluripotent stem cells (iPSCs) from fibroblasts of an SD patient with a lethal form of metatropic dysplasia,caused by a dominant mutation (I604M) in the calcium channel gene TRPV4. When micromasses were grown in chondrogenic differentiation conditions and compared with control iPSCs,mutant TRPV4-iPSCs showed significantly (Ptextless0.05) decreased expression by quantitative real-time polymerase chain reaction of COL2A1 (IIA and IIB forms),SOX9,Aggrecan,COL10A1,and RUNX2,all of which are cartilage growth plate markers. We found that stimulation with BMP2,but not TGF$\$1,up-regulated COL2A1 (IIA and IIB) and SOX9 gene expression,only in control iPSCs. COL2A1 (Collagen II) expression data were confirmed at the protein level by western blot and immunofluorescence microscopy. TRPV4-iPSCs showed only focal areas of Alcian blue stain for proteoglycans,while in control iPSCs the stain was seen throughout the micromass sample. Similar staining patterns were found in neonatal cartilage from control and patient samples. We also found that COL1A1 (Collagen I),a marker of osteogenic differentiation,was significantly (Ptextless0.05) up-regulated at the mRNA level in TRPV4-iPSCs when compared with the control,and confirmed at the protein level. Collagen I expression in the TRPV4 model also may correlate with abnormal staining patterns seen in patient tissues. This study demonstrates that an iPSC model can recapitulate normal chondrogenesis and that mutant TRPV4-iPSCs reflect molecular evidence of aberrant chondrogenic developmental processes,which could be used to design therapeutic approaches for disorders of cartilage. View Publication