Derivation of Induced Endothelial Cells

iECs were derived using a three-dimensional approach with modifications ^{10 (link)}. Briefly, to initiate differentiation, iPSCs were cultured in ultra-low, non-adhesive dishes to form embryoid body (EB) aggregates in EBM2 media (Lonza) in the absence of leukemia inhibitor factor (LIF). After 4 days of suspension culture, the EBs were reattached onto 0.2% gelatin-coated dishes and cultured in EBM2 medium supplemented with VEGF-A165 (50 ng/mL; PeproTech). After 3 weeks of differentiation, single cell suspensions were obtained using a cell dissociation buffer (Life Technologies) and labeled with APC-conjugated CD31 (eBiosciences) and PE-conjugated CD144 (BD Biosciences) anti-mouse antibodies. iECs were purified by fluorescence activated cell sorting (FACS) of CD31⁺CD144⁺ population. iECs were maintained in EBM2 media supplemented with recombinant murine vascular endothelial growth factor (50 ng/ml).

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de Almeida P.E., Meyer E.H., Kooreman N.G., Diecke S., Dey D., Sanchez-Freire V., Hu S., Ebert A., Odegaard J., Mordwinkin N., Brouwer T.P., Lo D., Montoro D., Longaker M.T., Negrin R.S, & Wu J.C. (2014). Transplanted Terminally Differentiated Induced Pluripotent Stem Cells Are Accepted By Immune Mechanisms Similar To Self-Tolerance. Nature communications, 5, 3903.

Publication 2014

EBM2 media VEGF-A165 cell dissociation buffer APC-conjugated CD31 PE-conjugated CD144

Anti antibodies Buffer Cell Cultured medium Dishes Embryoid body Gelatin Ipscs Leukemia Murine Vascular endothelial growth factor

Corresponding Organization :

Other organizations : Stanford University

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Variable analysis

independent variables

Three-dimensional approach with modifications
Culturing iPSCs in ultra-low, non-adhesive dishes to form embryoid body (EB) aggregates
Reattaching EBs onto 0.2% gelatin-coated dishes
Culturing in EBM2 medium supplemented with VEGF-A165 (50 ng/mL)

dependent variables

Differentiation of iPSCs into iECs
Percentage of CD31+CD144+ cells obtained by FACS

control variables

Absence of leukemia inhibitor factor (LIF) during EB formation
Maintaining iECs in EBM2 media supplemented with recombinant murine vascular endothelial growth factor (50 ng/ml)

controls

Positive control: None specified
Negative control: None specified

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