Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs. Six samples were analyzed. The gene expression profile of four iPS clones were compared to the H9 human embryonic stem cell line and the parent endothelial progenitor cell line.
Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.
Project description:Studies of miRNA profiling in early and late endothelial progenitor cells treated or not by cardioprotective nucleoside adenosine. Early outgrowth endothelial progenitor cells were obtained by adhesion of peripheral blood mononuclear cells of healthy volunteers. Late endothelial progenitor cells were obtained by purification of CD34+ peripheral blood cells and were cultured and amplified in endothelial-specific medium containing growth factors. Both cell types were treated by adenosine (10micromol/L) for 6 hours. Total RNA was extracted using mirVana Kit and quantified by Nanodrop. RNA was labeled and hybridized using Agilent miRNA Complete Labeling and Hyb Kit. 3 to 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Six independent experiments were performed.
Project description:Induced pluripotent stem cell (iPSC) technology allows for the generation of patient-specific pluripotent stem cells, from somatic cell sources, thereby providing a novel cell therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming and subsequent feasiblity of reprogramming into a pluripotent state. We used microarrays to detail the global gene expression profiles from blood cells. The use of blood cells allows for minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming, mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation. We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and mononuclear myeloid cells and tested the pluripotency of derived iPS clones.
Project description:Human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells differentiate into cells of the endothelial lineage, but derivation of cells with human umbilical cord blood endothelial colony forming cell (ECFC)-like properties has not been reported. Here we describe a novel serum- and stromal cell-free ECFC differentiation protocol for the derivation of clinically relevant numbers of ECFCs (> 108) from hiPS and hES cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, extensive replicative capacity, formation of human vessels that inosculated with host vasculature upon transplantation, but lacking in teratoma formation in vivo. We also identified NRP-1-VEGF165-KDR-mediated activation of KDR as a critical mechanism for the emergence and derivation of ECFCs from hiPS and hES cells. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Transcriptome sequencing of undifferentiated day 0 hiPS cells, Day 3 differentiated hiPS-derived mesoderm proginator cells, Day 12 hiPS-derived NRP-1+CD31+ cells, Day 12 H9-hES-derived NRP-1+CD31+ cells and cord blood-derived Endothelial colony forming cells.
Project description:Endothelial progenitor cells (EPCs) are circulating endothelial precursors shown to incorporate into foci of neovascularisation. Herein, we describe phenotypic characteristics of an EPC sub-type called endothelial colony-forming cells (ECFCs). Peripheral blood-isolated ECFCs expressed endothelial and progenitor surface antigens and displayed cobblestone-patterned colonies with clonal proliferative and angiogenic capacities in vitro. ECFCs demonstrated endothelial cell-like shear stress responses including cell alignment and PECAM-1 expression. Proteomic comparison with an endothelial reference population (human umbilical vein endothelial cells) confirmed a similar proteomic profile. Hierarchical clustering revealed two distinct ECFC clusters with differences in cell growth, proliferation and angiogenesis capacities. The cluster with compromised functionality was also associated with elevated blood pressure and impaired lipid profile. Our findings described an endothelial-like phenotype of blood-derived ECFCs with distinctive proteomic signatures based on cellular and clinical characteristics. ECFCs may aid in identifying novel mechanisms associated with cardiovascular disease risk and new targets to enhance angiogenesis.
Project description:Human induced pluripotent stem (iPS) cells derived from somatic cells of patients hold great promise for modelling human diseases. Dermal fibroblasts are frequently used for reprogramming, but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPS cells from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPS lines are comparable to human embryonic stem (ES) cells with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation and differentiation potential. Analysis of Immunoglobulin and T-cell receptor gene rearrangement revealed that some of the PBMC iPS cells were derived from T-cells, documenting derivation of iPS cells from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe and efficient way of generating patient-specific iPS cells. We isolated RNA from multiple human blood sources including peripheral blood mononuclear iPS cells, multiple somatic cells and iPS cells and human embryonic cells and hybridized them to Affymetrix gene expression microarrays.
Project description:Human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells differentiate into cells of the endothelial lineage, but derivation of cells with human umbilical cord blood endothelial colony forming cell (ECFC)-like properties has not been reported. Here we describe a novel serum- and stromal cell-free ECFC differentiation protocol for the derivation of clinically relevant numbers of ECFCs (> 108) from hiPS and hES cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, extensive replicative capacity, formation of human vessels that inosculated with host vasculature upon transplantation, but lacking in teratoma formation in vivo. We also identified NRP-1-VEGF165-KDR-mediated activation of KDR as a critical mechanism for the emergence and derivation of ECFCs from hiPS and hES cells. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders.
Project description:Human induced pluripotent stem (iPS) cells derived from somatic cells of patients hold great promise for modelling human diseases. Dermal fibroblasts are frequently used for reprogramming, but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPS cells from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPS lines are comparable to human embryonic stem (ES) cells with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation and differentiation potential. Analysis of Immunoglobulin and T-cell receptor gene rearrangement revealed that some of the PBMC iPS cells were derived from T-cells, documenting derivation of iPS cells from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe and efficient way of generating patient-specific iPS cells.
Project description:While others have reported that fetal liver contains a population of endothelial progenitors based on expression of cell surface markers or culture assays, this is the first proof of a CD31+Sca1+ progenitor by demonstrating highly efficient in vivo angiogenesis and a direct connection to the host vasculature. We have developed a novel isolation method based on collagenase digestion and culture on a fetal liver-derived feeder layer and demonstrate that the feeder cells or their supernatants are required for endothelial progenitor survival and proliferation. Proteogenomic profiling of the endothelial progenitors and the feeder cells was done with tandem mass spectrometry proteomics using MudPIT and gene transcript expression profiling using high density DNA microarrays. This approach identified a number of gene transcripts, proteins and candidate growth factor pathways that are likely to be involved in endothelial progenitor growth, differentiation and angiogenesis. Keywords = LEP - Liver Endothelial progenitor Keywords = Feeder Cells Keywords = Angiogenesis Keywords = Proteogenomics Keywords = Growth Factors Keywords = Fetal liver Keywords: parallel sample