Table 1.
Nucleotide sequences of human-specific primer sets.
Figure 1.
Expression of Cx43 in hESCs and hASCs.
The expression of endogenous Cx43 in hESCs and hASCs was confirmed by qRT-PCR (A) and Western blot analysis (B). Expression of OCT4 (red) and Cx43 (green) in hESCs and hASCs was observed by immunocytochemistry (C). Nuclei were counterstained with DAPI (blue). The arrow head indicates the expression of endogenous Cx43 in hESCs adjacent to hASCs (arrow). The white box indicates the Cx43 expression at the border line. All data are shown as the mean ± the standard deviation (SD) (n = 4; #, p>0.05; *, p<0.05). Scale bar, 100 μm.
Figure 2.
Functional gap junction channels were detected by the scrape loading/dye transfer assay for hASCs cultured alone and with hESCs. Phase contrast (A(a-c), B(a-c), C(a,b)), Lucifer yellow (A(d-f), B(d-f), C(c,d)) and rhodamine-dextran (A(g-i), B(g-i), C(e,f)) of hASCs and hESCs are shown. A scrape was made across the monolayer to allow the uptake of Lucifer yellow by the wounded cells. The dye was then transferred from the wounded cells to the adjacent, intact cells via functional gap junction channels. Lucifer yellow dye transfer was shown from hASCs to adjacent hASCs (A(e,f)); hESCs to adjacent hESCs (B(e,f); and hASCs to adjacent hESCs (C(c,d)). No-scrape group was employed as the normal conditions (A(d) and B(d)). Rhodamine-dextran was used as a negative control, showing no dye transfers from the wounded cells to neighbouring cells (A(h,i), B(h,i), C(e,f)). The black and white arrows indicate the scraped cells and the hESC colonies, respectively. The white dotted lines show the boundary between the hASCs and hESC colony. Scale bar, 100 μm.
Figure 3.
Effect of Cx43 downregulation on hESC stemness.
Downregulation of Cx43 and stemness gene (OCT4, SOX2, and NANOG) expression in Cx43-siRNA-treated hESCs was confirmed by qRT-PCR after 2 days culture (A) and Western blot analysis after 4 days culture (B). The percentage of AP-positive colonies formed by hESCs following Cx43-siRNA treatment was significantly lower than the percentage of AP-positive colonies formed by hESCs following control or scrambled-siRNA treatment (C). Immunocytochemical analysis demonstrated that OCT4 and Cx43 protein expression was reduced in Cx43-siRNA-treated hESCs versus control or scrambled-siRNA-treated hESCs (D). (C) and (D) were evaluated after 5 days culture. All data are shown as the mean ± the SD (n = 4; *, p<0.05). Scale bar, 100 μm.
Figure 4.
siRNA-mediated downregulation of Cx43 in hASCs.
The time-dependent, siRNA-mediated downregulation of Cx43 in hASCs was investigated by qRT-PCR (A) and Western blot analysis (B). Immunocytochemical analysis confirmed the Cx43 downregulation in hASCs after 3 days of siRNA treatment (C). Nuclei were counterstained with DAPI. All data are shown as the mean ± the SD (n = 4; *, p<0.05). Scale bar, 100 μm.
Figure 5.
Schematic diagram of Cx43-siRNA treatment method.
To downregulate Cx43 expression in hASC feeder cells, hASCs were seeded at 2.1×105 cells per 60 mm tissue culture plate before 24 h. hASCs were washed with PBS and then treated with scrambled-siRNA or Cx43-siRNA and cultured for 24 h. Twenty-four hours after transfection, hESCs clumps were transferred onto siRNA treated hASC feeder cells and maintained for 5 days.
Figure 6.
Culture of hESCs on Cx43 siRNA-treated hASCs.
Compared with treatment of hASCs with control or scrambled-siRNA, the Cx43-siRNA treatment of hASC feeder cells did not alter adhesion (A), colony growth (n = 20; B), gene expression (i.e., OCT4, SOX2, and NANOG) (C), AP-positive colony numbers (D), OCT4 expression (E), or chromosomal stability (F) of co-cultured hESCs. Nuclei were counterstained with DAPI (E). All data are shown as the mean ± the SD. (n = 4; #, p>0.05). Scale bar, 100 μm.