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INTRODUCTION The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. METHODS Expression of total YAP,its paralogue transcriptional co-activator with PDZ-binding motif (TAZ),and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis,C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally,YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. RESULTS YAP,but not TAZ,was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants,however,was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1,Id2 and Id3 in response to BMP-2. In developing mouse limbs,Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage,suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. CONCLUSIONS Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology. 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

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

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

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

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

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

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

Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However,despite the well-established role of EWS-FLI1 in tumor initiation,the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here,we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies,or collections of differentiating stem cells,generates cells with properties of Ewing sarcoma tumors,including characteristics of transformation. These cell lines exhibit anchorage-independent growth,a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore,these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth,which is a hallmark of Ewing sarcoma tumors.Oncogene advance online publication,12 October 2015; doi:10.1038/onc.2015.368. View Publication