技术资料

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs,NPs and neurons matured in culture for either 2 weeks (termed early neurons,E) or 4 weeks (termed late neurons,L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate,enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically,328 genes were differentially expressed between BPD and control L neurons including GAD1,glutamate decarboxylase 1 (2.5 fold) and SCN4B,the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology,GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism,protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD. View Publication

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability,which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans,making it important to minimize the levels of replication stress and telomere dysfunction. Here,the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A,exogenous nucleosides,or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast,adding exogenous nucleosides relieved dysfunction,suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However,because the loss of telomeres can lead to chromosome instability and cancer,we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction. 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

Despite major advances in the pathophysiological understanding of peripheral nerve damage,the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end,we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival,migration,and guide neurite extension. View Publication

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study,we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly,Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore,GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes,revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement,with initial repression of Nanog and Esrrb,then Sox2,and finally Oct4,alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes,suggesting that Gata6 functions as both a direct repressor and activator. Together,this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. View Publication

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening,biomarker discovery,and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here,we describe the (1) retroviral reprogramming strategies in the generation of human iPSC,(2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality,and (3) protocol of cardiac differentiation by modulation of Wnt signaling and $\$-catenin pathway. 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

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

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

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

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