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. 2024 Jun 9;9(6):e10690. doi: 10.1002/btm2.10690

FIGURE 2.

FIGURE 2

Homogeneous and stable expression of ChR2 in ChR2‐engineered hPSCs. (a) Relative expressions of ChR2 in ChR2‐engineered and control hPSCs. ChR2‐1, 2, 6, 9, 12, and 13, heterozygous clones; ChR2‐3, 4, 7, and 8, homozygous clones. Four technical replicates were evaluated for each line (n = 4). Significance was calculated using two‐tailed nested t‐test. (b) Western blot analyses of ChR2‐engineered hPSCs. ChR2‐2 and 9, heterozygous clones; ChR2‐3 and 4, homozygous clones; neg ctrl, negative control (non‐targeted hPSC); pos ctrl, positive control (HEK293 cells transfected with ChR2). (c) Immunofluorescence analysis for ChR2 in control hPSCs and ChR2‐engineered hPSC lines (ChR2‐2, ChR2‐3). Scale bar, 20 μm. (d) Quantification of relative ChR2 expression level in control hPSCs and ChR2‐engineered hPSC lines (ChR2‐2, ChR2‐3). Each dot represents the mean intensity of the ROI. Three ROIs from five colonies were analyzed (n = 15). Significance was calculated using an unpaired t‐test. (e) Relative expressions of ChR2 in ChR2‐engineered hPSC lines (ChR2‐2, ChR2‐3) following passages. Five technical replicates were evaluated in each group (n = 5). Significance was calculated using two‐tailed nested t‐test. (f) Immunofluorescence analysis of the pluripotency marker OCT4 in control hPSCs and ChR2‐engineered hPSC lines (ChR2‐2, ChR2‐3). Scale bar, 100 μm. (g) Immunofluorescence analysis of the pluripotency marker SOX2 in control hPSCs and ChR2‐engineered hPSC lines (ChR2‐2, ChR2‐3). Scale bar, 100 μm.