Fig. 4: Imaging of a cardiac reporter line confirms autofluorescence changes in cells under high differentiation efficiency conditions.
From: Label-free imaging for quality control of cardiomyocyte differentiation
NKX2.5EGFP/+ hPSCs were treated with 12 µM and 9 µM of CHIR99021 for the first 24 h to achieve low and high differentiation efficiencies, respectively. The NKX2.5EGFP/+ hPSC line expresses EGFP when the cardiac progenitor protein NKX2.5 is expressed, indicating that the cell has differentiated into CM, around differentiation day 7. a CM differentiation efficiencies were verified by flow cytometry on day 12 with cTnT labeling. Low differentiation efficiency (12 µM CHIR99021, left) and high differentiation efficiency (9 µM CHIR99021, right) are shown. Data were collected from three biological replicates and presented as mean ± SEM. Gating strategy to determine the percentage of cTnT positive population in hPSC-derived cells. Single-cell quantitative analysis of b NAD(P)H mean lifetimes (τm), c, d representative images, and e FAD mean lifetimes (τm), f, g representative images for low (0.3% cTnT+ on day 12) and high (84.1% cTnT+ on day 12) differentiation efficiencies on day 0 (“D0”, immediately pre-treatment) and day 1 (“D1”, 24 h post-treatment with CHIR99021), respectively. n = 1618, 1017 cells for 0.3% condition day 0; day 1, respectively. n = 1633, 1243 cells for 84.1% condition day 0; day 1, respectively. Data are presented as dot plots with bars for the mean and 95% CI. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests. ****p < 0.0001. h Single-cell quantitative analysis of NAD(P)H τm, τ2, α1, τ1 on differentiation day 8 (Differentiation efficiencies are indicated at the bottom as percent cTnT+ on day 12). Statistical significance was determined by Student’s T-test. ****p < 0.0001. n = 580 and 727 cells for 0.3% and 84.1% condition day 8, respectively. Data are presented as dot plots with bars for the mean and 95% CI. Representative images NAD(P)H τm and EGFP fluorescence in live cells for i low differentiation efficiency (0.3% cTnT+) and j high differentiation efficiency (84.1% cTnT+). ps, picoseconds. Source data are provided as a source data file.
