Results: 3

1.
Figure 1

Figure 1. Comparative analysis of hemangioblast development from independently-derived hESC lines.. From: Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines.

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.

Heather Melichar, et al. PLoS One. 2011;6(5):e19854.
2.
Figure 2

Figure 2. Skewed hematopoietic vs. endothelial potential from EB-derived CD34+CD45− cells.. From: Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines.

(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.

Heather Melichar, et al. PLoS One. 2011;6(5):e19854.
3.
Figure 3

Figure 3. hESC-derived hematopoietic progenitor cells are phenotypically and developmentally distinct from in vivo hematopoietic precursors.. From: Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines.

(A) Significant phenotypic differences among hESC-derived, cord blood, and fetal liver hematopoietic progenitor cells. Representative plots of cell surface expression of CD34, CD31, and CD45 on differentiated hESCs (EB culture for 9 days), and CD34+ cell enriched cord blood and fetal liver. (B) Relative number of lymphocyte committed cells (as evidenced by expression CD7) as compared to input population (CD34+ population enriched by FACS from day 9 EBs.) Three independent experiments are shown (Exp 1–3). The bottom panel denotes the average of the three independent data sets (Exp 1–3), after normalization to the fold change of the H1 sample in each experiment. Standard deviations between hESC cells with p<0.05 (*) are also indicated. (C) Analysis of CD7 and CD1a expression from FACS enriched EB-derived, fetal liver, or cord blood CD34+ cells differentiated on OP9-hDL1 co-cultures for 14 days. (D) The proportion of T lineage-committed cells was determined by flow cytometric analysis of CD7 (lymphocyte) and CD1a (T cell) expression. Duplicate experiments are shown as representative of differentiation of CD34+ cells into CD7+ cells in OP9-hDL1 co-culture conditions (Enriched CD34+ cells from HuES15, H9, and HSF6 hESC lines failed to expand/survive in co-culture with OP9-hDL1 and are thus not presented). (E) Representative flow plots of cell-surface CD4 and CD8 expression from fetal liver CD34+ subsets differentiated on OP9 (left) or OP9-hDL1 (center and right) cells for 28 days. Fetal liver cells expressing CD34 were sorted to >95% purity. Additionally, fetal liver subsets were also sorted on CD34+CD45lo or CD34+CD45hi populations due to a gradient of CD45 expression. Cells were co-cultured on either OP9 or OP9-DL1 as indicated. (F) Flow cytometric analysis of cell-surface CD43 staining on HuES8, HuES14, and H1-derived hematopoietic progenitors (EB culture, day 9) and human cord blood progenitors. Flow plots were gated on CD34+ cells.

Heather Melichar, et al. PLoS One. 2011;6(5):e19854.

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