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Derivation of endothelial colony forming cells from human pluripotent stem cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49074 | GEO | 2014/07/18

SECONDARY ACCESSION(S): PRJNA212913

REPOSITORIES: GEO

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Derivation of endothelial colony forming cells from human pluripotent stem cells

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.

2014-07-18 | E-GEOD-49074 | biostudies-arrayexpress

Global gene expression differences between blood- and lymphatic-specific endothelial colony forming cells

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DNA methylation profiling of cord blood progenitor endothelial cells from overweight and GDM pregnancies

Project description:Fetal progenitor endothelial cells (endothelial colony forming cells; ECFC) are recruited for repair, vascular growth and angiogenesis and their high abundance perinatally suggests a function in postnatal vasculogenesis and angiogenesis. In this study we profiled ECFCs from pregnancies of control, overweight and diabetic mothers to study if adverse pregnancies are associated with epigenetic variation in ECFCs.

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Distinct phenotype and proteomic signature of peripheral blood-endothelial colony-forming cells of subjects with varied cardiovascular disease risks

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.

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Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. Comparison of the expression patterns of ECFCs that were either cultured in FBS-containing medium or in serum-free medium (five replicates each).

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Project description:Background Endothelial cells are crucial for hemostasis as they produce Von Willebrand factor (VWF). Von Willebrand Disease (VWD) results from a deficiency or defects in VWF, However, diagnosis is difficult due to large differences between gene variants and bleeding phenotype. Here, we analyze the endothelial compartment of patients with VWD using Endothelial Colony Forming Cells (ECFCs). Methods 13 healthy controls and 10 patients were included and a total of 29 ECFC clones were derived. Plasma was analyzed and ECFC gene expression was measured by qPCR which was used to characterize clones. ECFCs were then morphologically and functionally tested by ELISA, imaging, scratch assay and mass spectrometry. Results VWF plasma levels and activity was reduced in patients, although values were higher than historically measured. Through hierarchical clustering of RNA expression profiles, we classified ECFCs into 2 clusters. Patients were matched with controls from the same cluster. Patient ECFCs did not show a uniform phenotype, but 3 of the 5 patients that did not respond to DDAVP showed clear retention of VWF in the endoplasmic reticulum. Furthermore, MS data showed proteomic clustering overlapping with RNA profiles and revealed a third group from lymphatic origin. An inverse protein correlation was seen between VWF levels and WPB count and cell area. No clear patient phenotype was observed but unique proteins were downregulated per clone. Conclusion This study investigates a broad panel of ECFCs derived from both healthy and diseased donors through comprehensive characterization using functional assays and proteomics. It emphasizes the current opportunities and challenges of utilizing ECFCs as a model to study WPB-specific mechanisms, while also providing an extensive overview of the molecular regulation of WPB machinery across endothelial cells and its functional implications.

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