技术资料

Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2,CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor $\$-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus,an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis,we generated iPSC-derived neurons from FOXG1(+/-) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/-) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains,where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-$\$1) were increased,whereas the levels of a number of excitatory synaptic markers (VGLUT1,GluA1,GluN1 and PSD-95) were decreased. In adult mice,GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/-) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses. View Publication

How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2),a homeobox transcription factor of msh family,as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs,while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs,and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore,MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription,while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly,SOX2 can promote the degradation of MSX2 protein,suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together,our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs. View Publication

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

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types,thus providing a platform for basic and clinical applications. However,pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here,we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival,self-renewal,and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. View Publication

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However,the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study,we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly,the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore,the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. View Publication

INTRODUCTION Numerous pathogenic mutations responsible for mitochondrial diseases have been identified in mitochondrial DNA (mtDNA)-encoded tRNA genes. In most cases,however,the detailed molecular pathomechanisms and cellular pathophysiology of these mtDNA mutations -how such genetic defects determine the variation and the severity of clinical symptoms in affected individuals- remain unclear. To investigate the molecular pathomechanisms and to realize in vitro recapitulation of mitochondrial diseases,intracellular mutant mtDNA proportions must always be considered. RESULTS We found a disease-causative mutation,m.5541CtextgreaterT heteroplasmy in MT-TW gene,in a patient exhibiting mitochondrial myopathy,encephalopathy,lactic acidosis,and stroke-like episodes (MELAS) with multiple organ involvement. We identified the intrinsic molecular pathomechanisms of m.5541CtextgreaterT. This mutation firstly disturbed the translation machinery of mitochondrial tRNA(Trp) and induced mitochondrial respiratory dysfunction,followed by severely injured mitochondrial homeostasis. We also demonstrated cell-type-specific disease phenotypes using patient-derived induced pluripotent stem cells (iPSCs) carrying ˜100 % mutant m.5541CtextgreaterT. Significant loss of terminally differentiated iPSC-derived neurons,but not their stem/progenitor cells,was detected most likely due to serious mitochondrial dysfunction triggered by m.5541CtextgreaterT; in contrast,m.5541CtextgreaterT did not apparently affect skeletal muscle development. CONCLUSIONS Our iPSC-based disease models would be widely available for understanding the definite" genotype-phenotype relationship of affected tissues and organs in various mitochondrial diseases caused by heteroplasmic mtDNA mutations� View Publication

Pluripotent stem cells,including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),have the potential to treat type 1 diabetes through cell replacement therapy. However,the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification,and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation,and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified,suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR,and the relationship between one of these miRNAs,miR-151a-5p,and its predicted target gene,SOX17,was investigated by luciferase assay,and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion,these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. 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

Tissue engineering strategies,based on solid/porous scaffolds,suffer from several limitations,such as ineffective vascularization,poor cell distribution and organization within scaffold,in addition to low final cell density,among others. Therefore,the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds,such as ideal chemical composition,the preservation of vascularization structure and perfect three-dimensional structure. In the present study,we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content,through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure,assessed after Hematoxylin and Eosin,as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis,performed,using SEM. Those essays allowed us to conclude that DM was indeed decellularized,with preserved extracellular matrix structure. Following characterization,decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected,but - to the best of our knowledge - never shown before,decellularization of murine heart matrices maintained matrix biocompatibility,as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though,heart DM presented a differentiation induction effect over those cells,which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed,even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed,in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices. View Publication

The liver is the main organ responsible for the modification,clearance,and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However,the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however,current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly,fetal hepatocytes acquire mature CYP450 expression only postpartum,suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid,a by-product of intestinal flora,activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes,while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive,permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells,compared to 0.62 for HepG2 cells. Finally,stem cell-derived hepatocytes demonstrate all toxicological endpoints examined,including steatosis,apoptosis,and cholestasis,when exposed to nine known hepatotoxins. CONCLUSION: Our work provides fresh insights into liver development,suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional,inducible,hPSC-derived hepatocyte for predictive toxicology. (Hepatology 2015). View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Current protocols for human pluripotent stem cell (hPSC) expansion require feeder cells or matrices from animal sources that have been the major obstacle to obtain clinical grade hPSCs due to safety issues,difficulty in quality control,and high expense. Thus,feeder-free,chemically defined synthetic platforms have been developed,but are mostly confined to typical polystyrene culture plates. Here,we report a chemically defined,material-independent,bio-inspired surface coating allowing for feeder-free expansion and maintenance of self-renewal and pluripotency of hPSCs on various polymer substrates and devices. Polydopamine (pDA)-mediated immobilization of vitronectin (VN) peptides results in surface functionalization of VN-dimer/pDA conjugates. The engineered surfaces facilitate adhesion,proliferation,and colony formation of hPSCs via enhanced focal adhesion,cell-cell interaction,and biophysical signals,providing a chemically defined,xeno-free culture system for clonal expansion and long-term maintenance of hPSCs. This surface engineering enables the application of clinically-relevant hPSCs to a variety of biomedical systems such as tissue-engineering scaffolds and medical devices. View Publication