Figure 2.

ADSC directed differentiation into corneal endothelial cells (CECs). Merge immunofluorescence images of the expression of S100 (red) and Na⁺/K⁺ ATPase (green) in ADSC cultures subjected to different protocols. Note S100 downregulation as demonstrated by lack of red fluorescence in every image (A): Ali protocol at 20 days of the second stage. (B): Wagoner protocol at 10 days of the second stage. (C): Quantitative RT-PCR of neural and corneal endothelial markers of ADSC-derived CEC cultures. Data are respective of the baseline expression in undifferentiated ADSCs (randomly assigned as 1). Note the significantly increased expression of Na⁺/K⁺ ATPase using both protocols and the decreased S100 expression. Comparisons can be made with human CECs directly isolated from donor corneas (d0) and cultured as usual for transplantation (d15). Moreover, compare the expression with induced pluripotent stem cells (iPSCs). (D): Na⁺/K⁺ ATPase-positive cells were detected at 20 days of the second stage using Zavan at first and then Ali in the second stage, also with S-100 downregulation (E): Na⁺/K⁺ ATPase-positive cells at 20 days of the second stage when using Jang at first and then Ali in the second stage, also with S-100 downregulation. Cell nuclei are stained with DAPI in blue. Bars represent 200 µm. * symbol indicates statistical significance difference compared to native ADSC. Δ symbol indicates statistical significance difference compared to human CECs directly isolated from donor corneas (d0). ■ symbol indicates statistical significance difference compared to human CECs cultured as usual for transplantation (d15). ○ symbol indicates statistical significance difference compared to ADSC directed differentiation into CECs with Ali protocol at 20 days of the second stage. ▼ symbol indicates statistical significance difference compared to ADSC directed differentiation into CECs with Wagoner protocol at 20 days of the second stage. (significance at p < 0.05).