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OBJECTIVE: Bone marrow-derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However,BMCs comprise a heterogeneous mixture of cells,and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4,we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs. METHODS AND RESULTS: Human CXCR4(+) and CXCR4(-) BMCs were sorted by magnetic beads. CXCR4(+) BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4(+) and CXCR4(-) BMCs. Injection of CXCR4(+) BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4(-) BMCs. Likewise,capillary density was significantly increased in CXCR4(+) BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors,we analyzed BMC supernatants for secreted factors. Importantly,supernatants of CXCR4(+) BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB. CONCLUSIONS: CXCR4(+) BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically,their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes,indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model,transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (ptextless0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition,transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease. 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

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for textgreater10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media,the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR),HEScGRO,and KnockOut media,mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions,cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4,stage-specific embryonic antigens 4 (SSEA-4),and Tra-1-60. In addition,hESC maintained the expression of podocalyxin-like protein-1 (PODXL),an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills textgreater90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies,derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal,endodermal,and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%,higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture,it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition,we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted,aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis,aligned collagen substrates choreographed ordered matrix mineralization. Likewise,myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures. View Publication

The multidrug transporter ABCG2 in cell membranes enables various stem cells and cancer cells to efflux chemicals,including the fluorescent dye Hoechst 33342. The Hoechst(-) cells can be sorted out as a side population with stem cell properties. Abcg2 expression in mouse embryonic stem cells (ESCs) reduces accumulation of DNA-damaging metabolites in the cells,which helps prevent cell differentiation. Surprisingly,we found that human ESCs do not express ABCG2 and cannot efflux Hoechst. In contrast,trophoblasts and neural epithelial cells derived from human ESCs are ABCG2(+) and Hoechst(-). Human ESCs ectopically expressing ABCG2 become Hoechst(-),more tolerant of toxicity of mitoxantrone,a substrate of ABCG2,and more capable of self-renewal in basic fibroblast growth factor (bFGF)-free condition than control cells. However,Hoechst(low) cells sorted as a small subpopulation from human ESCs express lower levels of pluripotency markers than the Hoechst(high) cells. Similar results were observed with human induced pluripotent stem cells. Conversely,mouse ESCs are Abcg2(+) and mouse trophoblasts,Abcg2(-). Thus,absence of ABCG2 is a novel feature of human pluripotent stem cells,which distinguishes them from many other stem cells including mouse ESCs,and may be a reason why they are sensitive to suboptimal culture conditions. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

Up to now,no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes,which hampers its application in gene therapy and immunotherapy areas. Here,we report that LVs incorporating measles virus (MV) glycoproteins,H and F,on their surface allowed transduction of 50% of quiescent B cells,which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover,the naive and memory phenotypes of transduced resting B cells were maintained. Importantly,H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells,B-cell chronic lymphocytic leukemia cells,blocked in G(0)/G(1) early phase of the cell cycle. Thus,H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells. View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

Magnetic resonance imaging (MRI) may emerge as an ideal non-invasive imaging modality to monitor stem cell therapy in the failing heart. This imaging modality generates any arbitrary tomographic view at high spatial and temporal resolution with exquisite intrinsic tissue contrast. This capability enables robust evaluation of both the cardiac anatomy and function. Traditionally,superparamagnetic iron oxide nanoparticle (SPIO) has been widely used for cellular MRI due to SPIO's ability to enhance sensitivity of MRI by inducing remarkable hypointense,negative signal,blooming effect" on T2*-weighted MRI acquisition. Recently� View Publication