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The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2),a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly,whereas normal HSCs were rapidly exhausted after serial transplantations,overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast,similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a genetic genomics" screen� View Publication

With the advent of induced pluripotent stem cells and directed differentiation techniques,it is now feasible to derive individual-specific cardiac cells for human heart tissue engineering. Here we report the generation of functional engineered human cardiac patches using human induced pluripotent stem cells-derived cardiac cells and decellularized natural heart ECM as scaffolds. The engineered human cardiac patches can be tailored to any desired size and shape and exhibited normal contractile and electrical physiology in vitro. Further,when patching on the infarct area,these patches improved heart function of rats with acute myocardial infarction in vivo. These engineered human cardiac patches can be of great value for normal and disease-specific heart tissue engineering,drug screening,and meet the demands for individual-specific heart tissues for personalized regenerative therapy of myocardial damages in the future. View Publication

Human cell transformation is a key step for oncogenic development,which involves multiple pathways; however,the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC),we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung,breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and,OCT4,in turn,stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover,we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a,promotes p63 but suppresses p53 expression,which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and,most likely,also to the iPSC process. View Publication

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here,we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions,resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions,which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. View Publication

The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics,force generation,and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness,we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly,our approach has the potential for broad application in the study of cardiac disease,drug discovery,and cardiotoxicity screening. View Publication

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC,from both human and murine origin,has been reported in many studies. MSC transformation depends on the culture conditions,the origin of the cells and the time on culture; however,the precise biological characteristics involved in this process have not been fully defined yet. In this study,we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate,a shorter doubling time and also morphologic and phenotypic changes,as compared to MSC derived from wild-type animals. Furthermore,the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition,not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover,genomic instability,changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition,the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice,the Cre/loxP system was used,and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However,double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation,causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes,mast cells,and eosinophils,but not in neutrophils,lymphocytes,or nonhematopoietic tissues. In summary,GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time,mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins,including SMAD1. In previous work,we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly,we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely,when Smad1 is depleted in FlK1(+) mesoderm,at a stage after hemangioblast commitment,the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis,including Gata1,Runx1 and Eklf,rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity,indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism,hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore,this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors. View Publication

Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine,but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here,we determined that the downregulation of miR-302 suppresses the teratoma formation,hampers the self-renewal and pluripotency,and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1,which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly,we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation. View Publication