Comparative analysis of hemangioblast development from independently-derived hESC lines.

<p>Several hESC lines were differentiated as embryoid bodies (EB) for nine days after several trypsin passages (A, top panel) or after several manual passages (A, middle panel) in EB media without lineage-skewing cytokines. CD34⁺ and CD34⁺CD45⁺ development was determined by flow cytometric analysis of several cell surface markers indicative of differentiation state. The proportion of hESC-derived CD34⁺CD45⁻ cells is presented on differentiating hESCs in black. The proportion of CD34⁺CD45⁺ progenitors is indicated in white. HuES8, HuES14, and HuES15 cell lines were highly susceptible to gross karyotypic abnormalities during trypsin passage (as indicated). H1, H9, and HSF6 manually passaged cells had previously been passaged with trypsin (>5 manual passages before differentiation). (A, bottom panel) Independently-derived hESC were differentiated on an OP9 monolayer for nine days, and CD34 and CD45 cell surface expression analyzed by flow cytometry. Two representative experiments of each condition are presented. (B) Abnormal karyotypes observed in trypsin passaged cells. (C) Representative time course of CD34 expression on manually passaged, independently-derived hESCs differentiated as EBs or on an OP9 monolayer. CD34 expression was analyzed on days 3, 6, 9, 12, 15, 18.</p

Skewed hematopoietic vs. endothelial potential from EB-derived CD34⁺CD45⁻ cells.

<p>(A) The indicated hESC lines were first differentiated as EBs in EB media without lineage skewing cytokines. CD34⁺CD45⁻ cells were enriched by fluorescence activated cell sorting (FACS) on day 10 of EB culture and differentiated on fibronectin-coated plates in the presence of IL-3, IL-6, SCF, G-CSF, Flt3L, and BMP4 for an additional 7 days. Representative flow cytometry plots of CD45 (hematopoietic marker) and VE-cadherin (endothelial marker) are presented. (B) Comparative analysis of endothelial potential from independently-derived hESC lines. Endothelial differentiation of hESC lines was determined by a two-step culture. hESCs were initially differentiated for 9 days as EBs as in (A), and CD34⁺ cells enriched by FACS. CD34⁺ cells were plated on fibronectin-coated plates in the presence of an endothelial growth factor cocktail containing bovine pituitary extract, heparin, and hVEGF, and analyzed after 7 additional days in culture. Representative flow cytometry plots of CD45 and VE-cadherin are presented. (C) Graphs depict the relative number of hematopoietic (CD45⁺) or (D) endothelial lineage (VE-cadherin⁺) cells as identified by flow cytometry over the starting (CD34⁺, CD45⁻, VE-cadherin⁻) population. Three independent experiments are shown in (C) and (D). The right panels denote the average of the three independent data sets with error bars and standard deviation between hESC lines. * denotes p<0.05.</p