Further characterization of a new in vitro angiogenesis model under serum free culture conditions; suppression of endothelial cell differentiation by serum

Abstract Embryonic stem (ES) cells can differentiate into many different somatic cells in culture. To better correlate hematopoietic and endothelial cell differentiation of ES cells in currently available protocols, we compared fetal liver kinase-1 (Flk-1)–, stem cell leukemia (Scl)–, and vascular endothelial–cadherin (VE-cadherin)–expressing cells generated in embryoid bodies (EBs) and on OP9 cells. We report that the kinetics of Scl and Flk-1 expression were similar in EBs and OP9 cells, although Flk-1 expression was extended on OP9 cells. CD45+ and Ter-119+ cells developed more efficiently in EBs, whereas VE-cadherin+ cells developed largely on OP9 cells. Cell sorting and replating studies showed that Scl+ cells, not Flk-1+ or VE-cadherin+ cells, were enriched for primitive and definitive hematopoietic progenitors. Our studies indicate that optimal hematopoietic and endothelial cell differentiation occur in EBs and on OP9 cells, respectively. Regardless of the culture systems used, Scl is the most relevant marker for enriching primitive and definitive hematopoietic progenitors.

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Abstract 12495: Fibrin-Thrombin Patch for Endothelial Cell Differentiation and Cardiac-Tissue Patch Manufacturing Using Human Induced-Pluripotent Stem Cells

Circulation ◽

10.1161/circ.130.suppl_2.12495 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Sophia Zhang ◽

James Dutton ◽

Liping Su ◽

Jianyi Zhang ◽

Lei Ye

Keyword(s):

Stem Cells ◽

Endothelial Cell ◽

Cell Differentiation ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cardiac Tissue ◽

Differentiation Protocol ◽

Endothelial Cell Differentiation ◽

Induced Pluripotent

Aim: To explore the feasibility using fibrin-thrombin patch for endothelial cell differentiation and cardiac scaffold manufacturing using cardiac cells derived from human induced-pluripotent stem cells (hiPSCs). Method and Result: hiPSCs were dissociated into single cells and seeded into three-dimensional (3D) fibrin-thrombin patches and undergo a two-stage differentiation protocol. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. A human cardiac-tissue patch (hiCP) was developed by seeding the hiPSC-ECs with hiPSC-derived cardiomyocytes (hiPSC-CMs) and smooth muscle cells (hiPSC-SMCs) into a 3D fibrin scaffold. The hiCP began to contract 3 days after synthesis, 4 days earlier than patches that were created identically but without hiPSC-ECs, and continued to beat regularly (100-120 beats/min) for at least 4 weeks in vitro. Conclusion: These data demonstrate that this new 3D differentiation protocol can efficiently generate stable ECs from hiPSCs and, furthermore, that the differentiated hiPSC-ECs can be combined with hiPSC-CMs and -SMCs to construct an hiCP with improved contractile activity. Our observations also suggest that interactions between the cardiac endothelium and myocytes may contribute to the optimized beating of hiCPs.

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