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The mechanisms underlying human germ cell development are largely unknown,partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here,we studied NANOS3 and DAZL,which have critical roles in germ cell development in several species,via their over expression in human embryonic stem cells using global transcriptional analysis,in vitro germ cell differentiation,and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition,we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL,our results suggest a post-transcriptional regulation mechanism in hES cells. In addition,we found that DAZL suppressed the translation of OCT4,and affected the transcription of several genes associated with germ cells,cell cycle arrest,and cell migration. Furthermore,DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. View Publication

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease,as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture,differentiation,isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d),isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes,respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases,inflammatory diseases,etc.,due to their pleiotropic properties. However,largely incongruent data were obtained from different MSC-based clinical trials,which may be partially due to functional heterogeneity among MSC. Here,we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP),which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression,proliferation,migration,multipotency,immunomodulatory activity and global gene expression profile. Moreover,the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC),and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly,NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo,as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies. View Publication

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness,a process termed licensing or education,but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept,we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog,TMIGD2) and 2B4 (CD244,SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody,the CD28H transmembrane domain,and the fusion of CD28H,2B4,and TCR$\zeta$ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCR$\zeta$ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore,such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore,engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies,such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing. View Publication

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 microM fumonisin B1,a fungal inhibitor of mammalian ceramide synthase,inhibited incorporation of [3H]galactose/glucosamine and [14C]-serine into complex sphingolipids of cultured cerebellar neurons. Under this condition,the expression of Purkinje cell-enriched sphingolipids,including GD1 alpha,9-O-acetylated LD1 and GD3,and sphingomyelin,was significantly decreased. After 2 weeks' exposure to fumonisin B1,dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag,but their branches began to degenerate. In some cells,formation of elongated dendrite trees was severely impaired. However,treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells,morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed,these effects of fumonisin B1 were reversed,but not completely,by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino dcaproyl]sphingosine (C6-NBD-ceramide),a synthetic derivative of ceramide. Thus,we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells. View Publication

Neurogenesis occurs continuously in two brain regions of adult mammals,underpinned by a pool of resident neural stem cells (NSCs) that can differentiate into all neural cell types. To advance our understanding of NSC function and to develop therapeutic and diagnostic approaches,it is important to accurately identify and enrich for NSCs. There are no definitive markers for the identification and enrichment of NSCs present in the mouse brain. Recently,a fluorescent rosamine dye,CDy1,has been identified as a label for pluripotency in cultured human embryonic and induced pluripotent stem cells. As similar cellular characteristics may enable the uptake and retention of CDy1 by other stem cell populations,we hypothesized that this dye may also enrich for primary NSCs from the mouse brain. Because the subventricular zone (SVZ) and the hippocampus represent brain regions that are highly enriched for NSCs in adult mammals,we sampled cells from these areas to test this hypothesis. These experiments revealed that CDy1 staining indeed allows for enrichment and selection of all neurosphere-forming cells from both the SVZ and the hippocampus. We next examined the effectiveness of CDy1 to select for NSCs derived from the SVZ of aged animals,where the total pool of NSCs present is significantly lower than in young animals. We found that CDy1 effectively labels the NSCs in adult and aged animals as assessed by the neurosphere assay and reflects the numbers of NSCs present in aged animals. CDy1,therefore,appears to be a novel marker for enrichment of NSCs in primary brain tissue preparations. View Publication

Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study,we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs. Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR. Key findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers,generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers,followed by early cardiac committed markers,structural and ion channel genes. Furthermore,our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin,actinin,myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally,microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol,reduced Na+ influx with quinidine,prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil. Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine. textcopyright 2012 Elsevier Inc. All rights reserved. View Publication

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-β[2-(di-n-butylamino)-6-naphthyl]butadienylquinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses,we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision,generating nearly identical values for AP duration (AP durations at 10%,50%,and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure,with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally,di-4-ANBDQBS allowed for moderate-throughput analyses,increasing throughput textgreater10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. View Publication