Figure 2.

Differentiation of TkDN4-M into Human Intestinal Organoids

(A) Schematic procedures for intestinal organoid differentiation from human iPSC lines.

(B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm.

(C) Magnified images of the spheroids and organoids. Scale bars, 40 μm.

(D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ^∗p < 0.05, Student's t test.

(E) Relative mRNA levels of SOX17 and CXCR4 in TkDN4-M cells differentiated into spheroids in the presence or absence of WNT3A/FGF2 supplementation were determined by qPCR and normalized to HPRT. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ^∗p < 0.05, Student's t test.

(F) Relative mRNA levels of the indicated genes in TkDN4-M cells during the course of differentiation (days 0, 3, and 7 [spheroids], and organoids) were determined by qPCR and normalized to HPRT. The highest expression levels of each gene in each differentiation stage were considered as “1.” The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM.

(G) Images of TkDN4-M-derived organoids on day 7 after culture in 25% WRN CM, or the corresponding recombinant proteins. Scale bars, 200 μm.

(H) Relative mRNA levels of LGR5 and VIL1 in TkDN4-M organoids cultured for short-term (passage 19) and long-term (passage 53) were determined by qPCR and normalized to HPRT. The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM.

(I) Whole-mount images of TkDN4-M-derived organoids stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm.