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

Fig. 4.

Fig. 4.

The downregulation of Hath6 in both H9 and H1 hESCs inhibits the development of endothelial characteristics during differentiation. (A) The downregulation of Hath6 in H9 hESCs does not affect the expression of pluripotency markers, including Oct4, Sox2 and Nanog (n = 3). (B–F) Results from the stepwise induction of H9 hESCs from the end of stage two. (B) The expression of Hath6 and endothelial-related genes, including eNOS, CD34, CD31, KDR and VE-cadherin, in shHath6-H9-hESC-EPCs was significantly downregulated compared with the mock control (n = 6). (C–E) FACS analyses of CD31 and KDR expression at H9-hESC differentiation stage two. The numbers indicate a decreased percentage of VE-cadherin+ cells (C), KDR+ cells and CD31+KDR+ cells (D) due to the downregulation of Hath6. (E) Statistical analysis shows a significant difference in these cells compared with the control (n = 6). (F) The angiogenesis assay and the counting of closed tube-like structures per visual field (n = 3) revealed that siHath6-H9-EPCs cannot form tube-like structures on Matrigel, in contrast to the control group. Scale bar: 200 µm. (G–I) Results of H1 hESC stage two induction. (G) RT-PCR (n = 6) and (H) FACS analyses indicate that the downregulation of endothelial-specific genes correlated with the knockdown of the Hath6 gene. (I) The statistical analysis showed a significant difference in these cells compared with the mock control (n = 3). (J–L) Results of H9 hESC stage three induction. (J) RT-PCR (n = 6) and (K) FACS analyses show the downregulation of endothelial genes in sh-Hath6-H9-hESCs compared with the mock control. The RT-PCR analysis shows that the endothelial-specific genes, with the exception of VE-cadherin, are significantly decreased compared to the control. (L) The uptake of DiI–Ac-LDL by sh-Hath6-hESCs at stage three was lower than that observed in sh-mock-hESCs. The arrows indicate cells that have taken up DiI–Ac-LDL. Scale bar: 100 µm. (M) Results of H1 hESC stage three induction. After culture in EGM2 medium for 6 days, FACS analysis indicates that most of the hESC derivations are CD45-negative. The downregulation of Hath6 decreases the percentage of CD45−CD31+KDR+ cells, CD45−CD31+VE-cadherin+ cells and total CD31+ cells, as determined by FACS analysis. *P<0.05, **P<0.01. In panels C,K,M the red and blue lines indicate the IgG isotype control and specific antibody staining, respectively.

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