Figure 3.

HEB^−/− hESCs Display Defects in Mesoendodermal Induction and Early Hematopoietic Differentiation

(A) Experimental overview of embryoid body (EB) formation and differentiation. BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; IL, interleukin; EPO, erythropoietin; SCF, stem cell factor; IGF1, insulin-like growth factor 1; FLT3L, FMS-like tyrosine kinase 3 ligand; TPO, thrombopoietin.

(B) Reverse-transcriptase PCR analysis of HEB transcript (HEBCan, canonical; HEBAlt, alternative) expression at various stages of EB differentiation, and in sorted day-8 (d8) CD34⁺ cells (last column). GAPDH was measured as a loading control.

(C) qRT-PCR analysis for the expression of pluripotency and differentiation markers in undifferentiated hESCs (day 0 [d0]) versus d4 EB-derived cells.

(D) Flow-cytometric analysis of CD34 and KDR, CD144, and CD31 expression on d8 EB-derived cells.

(E and F) Percentages (E) and numbers (F) of CD34⁺ cells in d8 EBs.

(G) qRT-PCR analysis of the expression of mesodermal and hematopoietic genes in CD34⁺ cells. For qRT-PCR graphs, mRNA levels are shown relative to GAPDH.

Error bars represent mean ± SD (n = 3 independent experiments). ^∗∗p < 0.01; ^∗∗∗p < 0.005 by Student's t test. Images in (B) and plots in (D) are representative of three independent experiments. See also Figure S4.