Figure 1.

Endothelial Differentiation of the Clinical-Grade hESC Line RC11

Differentiated cells analyzed on day 8 of the protocol predominantly co-expressed the endothelial markers CD31 and CD144 with few, if any, detectable residual pluripotent hESCs. (A) Representative flow cytometric analysis for the endothelial (left panels) and pluripotent markers (middle and right panels) with the appropriate isotype controls is shown. Cells were pre-gated for viable cells (FSC/SSC; 10,000 events) and doublet exclusion (FSC-A/FSC-H). (B) Day 8 hESC-ECP characteristics assessed against a target profile determined at the start of the study are shown; n = 21 replicates. (C) qPCR-detected expression of selected pluripotent (NANOG, OCT4, and SOX2) and endothelial (CD31, KDR, and CD34) genes in differentiated RC11 cells shows the downregulation of pluripotency and acquisition of endothelial phenotype in comparison to mRNA from human umbilical vein endothelial cells (HUVECs) as a positive control. Data are shown as 2ΔCt × 1,000 compared to the housekeeping gene β-actin. hESC data are n = 4 biological replicates assayed in triplicate, HUVEC n = 3 in triplicate; *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001 denote significance compared to d0; †p < 0.05, ††p ≤ 0.01, and †††p ≤ 0.001 denote level of significance compared to HUVECs using one-way ANOVA with Tukey’s post hoc test. All data represent mean ± SEM.