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. 2014 Apr 1;127(7):1428–1440. doi: 10.1242/jcs.136358

Fig. 2.

Fig. 2.

Endogenous expression of Hath6 during hESC differentiation. (A) Morphology of differentiated hESCs on days 0, 6 and 10 of embryoid body (EB) formation. Scale bar: 200 µm. (B) The real-time PCR results show the upregulation of Hath6, as well as endothelial-related genes, including CD31, KDR, VE-cadherin and ETV2, during the spontaneous differentiation of hESCs into EBs. Each data point represents the average of six experiments. Gene expression was first normalized to the expression of the housekeeping gene GAPDH. The fold induction of each tested gene was then normalized to the expression in undifferentiated hESCs. The data represent the means±s.d., n = 6. (C) Schematic of a three-step hESC-EC induction system. (D) Immunofluorescence staining observed by confocal microscopy indicates the co-expression of Hath6 (red) and the endothelial cell marker CD31 (green) in cells at stage two of hESC differentiation. Scale bars: 50 µm. A magnified view of the co-expression of Hath6 and CD31 is shown in the second panel from the right. Scale bar: 20 µm. The images are representative of three independent experiments. (E) Expression of Hath6 during endothelial differentiation. The RT-PCR results illustrate the increasing expression of Hath6, eNOS, CD31, VE-cadherin and ETV2 during phase differentiation, especially at stage three (n = 6). The expression of each tested gene was normalized to the expression in undifferentiated hESCs. The highest expression levels of CD34 and KDR in our differentiation system were observed at stage two. The data represent the means±s.d. Compared with undifferentiated hESCs, none of the genes shows a significant difference in expression in the stage one cells. Compared with the results for stage one, all of the genes in the stage two cells show a significant increase in expression (P<0.05). Compared with the results for stage two, all of the genes except KDR show a significant increase in expression at stage three (P<0.05). (F) Hath6 is enriched in CD31+KDR+ endothelial progenitor cells isolated from stage two hESC derivations (2.6-fold, **P<0.01).

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