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AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy,particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity,critical elements for their survival and differentiation,have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and,more generally,to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes,a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand,A-769662,a well-characterized AMPK activator,was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice,providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation,we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved,whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation,p27 being involved in this regulation. View Publication

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model. View Publication

Hybridomas producing mouse monoclonal antibodies to antigens of the human mammary carcinoma cell line,MCF-7,have been generated by electric field-mediated fusion at a frequency ten times higher than by polyethylene glycol. One of the monoclonal antibodies obtained recognizes a cytoskeletal structure restricted to epithelial cells and carcinomas with a distribution pattern resembling cytokeratin 19. View Publication

Background Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling,cardiotoxicity screening,drug discovery,and cell-based therapies. Despite the fundamental importance of Ca2+-induced Ca2+ release in excitation-contraction coupling,the mechanistic basis of Ca2+ handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. Objectives To study Ca2+ sparks as unitary events of Ca2+ handling for mechanistic insights. Methods To avoid ambiguities owing to the heterogeneous nature,we experimented with hESC-VCMs,purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction. Results Ca2+ sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine,tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model,we further showed that RyRs could be sensitized by Ca2+. Increasing external Ca2+ dramatically escalated the basal Ca2+ and spark frequency. Furthermore,RyR-mediated Ca2+ release sensitized nearby RyRs,leading to compound Ca2+ sparks. Depolarization or L-type Ca2+ channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca2+-dependent cytosolic Ca2+ increase and reduced the sarcoplasmic reticulum content. By contrast,removal of external Na+ or the addition of the Na+-Ca2+ exchanger inhibitor (KB-R7943 and SN-6) had no effect,suggesting that the Na+-Ca2+ exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca2+ uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca2+ waves. Conclusion Taken collectively,our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca2+-induced Ca2+ release. However,the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development. View Publication

Summary Rett syndrome (RTT) is caused by mutations of MECP2,a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show,using an isogenic human embryonic stem cell model of RTT,that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells. View Publication

Human embryonic stem cells (hESCs),due to their self-renewal capacity and pluripotency,have become a potential source of transplantable $\$-cells for the treatment of diabetes. However,it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study,we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers,had the ability to differentiate into three germ layers,and maintained a normal karyotype after 10 passages of subculture. Next,we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover,we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus,we provided a totally defined condition from hESC culture to insulin-producing cell differentiation,and the derived cells could be a therapeutic resource for diabetic patients in the future. View Publication

How tumor suppressor p53 selectively responds to specific signals,especially in normal cells,is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation,induced by retinoic acid,versus DNA damage,caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and,in pluripotent hESCs,are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases,UTX and JMJD3,to chromatin. In contrast,genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation,a process highly distinct from stress-induced p53 response in hESCs. View Publication

Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species,the signaling pathways that control this transition remain poorly understood. Here,we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential,whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators,whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively,these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells. View Publication

Alloreactivity negatively influences outcomes of organ transplantation or HCT from allogeneic donors. Standard pharmacologic immune suppression impairs T-cell function and jeopardizes the beneficial reconstitution of Tregs. Murine transplantation models have shown that STAT3 is highly expressed in alloreactive T cells and may be therapeutically targeted. The influence and effects of STAT3 neutralization in human alloreactivity,however,remain to be elucidated. In this study,S3I-201,a selective small-molecule inhibitor of STAT3,suppressed human DC-allosensitized T-cell proliferation and abrogated Th17 responses. STAT3 blockade significantly enhanced the expansion of potent iTregs and permitted CD8(+) cytolytic effector function. Mechanistically,S3I-201 polarized the ratio of STAT phosphorylation in favor of STAT5 over STAT3 and also achieved a significant degree of Foxp3 demethylation among the iTregs. Conversely,selective impairment of STAT5 phosphorylation with CAS 285986-31-4 markedly reduced iTregs. STAT3 represents a relevant target for achieving control over human alloresponses,where its suppression facilitates STAT5-mediated iTreg growth and function. View Publication

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However,the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here,we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition,manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally,analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively,our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs. ?? 2013 The Authors. View Publication

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways,mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts,from several donors,into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2,CLSTN1,ATP2A1,PALM,ITGA6,KIF13A,FMNL3,PPIP5K1,MARK2 and FNIP1),implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks. View Publication