Fig. 1

Derivation of CHIR99021-induced HE and iPSC-SR2 characterization. a Phenotypic characterization of iPSC-SR2: phase-contrast image of the cells when grown on MEFs. Expression of alkaline phosphatase (AP, red) and of characteristic pluripotency genes (TRA 1-80 (green), Nanog (red), and TRA 1-60 (green) and OCT4 (red) overlay) assessed by immunostaining. b Timeline of lympho-myloid differentiation and representative phase-contrast image of hPSCs differentiating to vascular progenitors on day 5 after induction with the small molecule CHIR99021. c Induction efficiency of CHIR99021-induced vs OP9 cocultured pluripotent cells as a percentage of CD31⁺ cells. Error bars are SEM of three independent experiments. d Homogeneity of CD31⁺ double-positive cells obtained from CHIR99021 induction vs heterogeneous population obtained from OP9 coculture. e At day 5, CD31⁺ cells were enriched with MACS selection column and quantified by flow cytometry for CD31 and CD144 expression. Phenotypic and functional characterization of isolated cells: phase-contrast image of cells when grown in endothelial medium, tube formation assay with calcein AM staining, expression of vWF assessed by immunostaining (red) overlay with Dapi, and expression of VE-cadherin assessed by immunostaining. f Cytometric flow analysis demonstrating homogeneity of CD31⁺ and CD144 coexpression by vascular cells at passage 3. D day, hPSC human pluripotent stem cell, vWF von Willebrand factor