FIGURE 3.

Characterization of endoderm induction (ED). (A) Differentiation protocol for the induction of lung epithelium from human IPSCs. After 24 h in static cultures, cells were differentiated in four stages spanning 20 days of culture. (B) On day four, qRT-PCR of endoderm-associated markers CXCR4, FOXA2, and pluripotency marker OCT3/4 was performed. (C) Gene expression and immunohistochemistry analyses conducted on day 6 and representative immunohistochemistry images showing Nkx2.1, on day 6 in static (II), dynamic (III), and 2D culture (IV) cultures. Scale bar, 20 µm. Immunohistochemistry controls are presented in Supplementary Figures S1, 2. Error bars show the mean fold change on day 4 and day 6, error bars show one standard deviation, n = 3 biological replicates. Gene expression was normalized to day 0 (2^−ΔΔCT method). p-values were obtained using one-way ANOVA, where ****p ≤ 0.0001, ***p ≤ 0.001, and **p ≤ 0.01. IPSC, induced pluripotent stem cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; CXCR4, C-X-C chemokine receptor 4; FOXA2, forehead box A2; RI, rock inhibitor; ANOVA, analysis of variance.