Fig. 1. Stepwise protocol for generating colonic organoids. (A) Schematic diagram showing the modification of previously reported protocols for colonic organoids, with or without bone morphogenetic protein (BMP) signaling stimulation. (B) Decrease in pluripotency markers OCT4 and NANOG during differentiation process. (C) Expression level of mRNA for SOX17 and FOXA2 on day 3 of differentiation towards definitive endoderm (DE). (D) Immunoblot images of protein expression for SOX17 and FOXA2 on the differentiation stage of DE. GAPDH was used as an internal control. (E) Cells expressing a protein for SOX17 and FOXA2 on the DE stage. (F) Counting results for SOX17 and FOXA2 expressing cells from (E). (G) Expression level of mRNA for CDX1 and KLF5 on day 7 of differentiation towards hindgut (HG) cells. (H) Immunoblot images of protein expression for CDX1 and KLF5 on the differentiation stage of hindgut. GAPDH was used as an internal control. (I) Cells expressing a protein for CDX1 and KLF5 on the hindgut stage. (J) Counting results for CDX1 and KLF5 expressing cells from (I). (K) Schematic diagram showing the embedding process of hindgut cells that formed clusters into an artificial ECM matrix in clump form. (L) BMP4 stimulation during the embedding stage increases expression of hindgut hox genes responsible for posterior development. (M and N) Increase in mRNA and protein expression of colon-restricted chromatin factor SATB2 with BMP4 application. (O) Identification of a cell cluster expressing the SATB2 protein in the inner center of the colonic organoid. Data expressed as mean±SD (n=9, biological repeat). p-values calculated by one-way ANOVA (B, L, M) or unpaired t-test (C, F, G, and J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. All scale bars=50 μm. DE: definitive endoderm, HG: hindgut, CO: colonic organoid.

International Journal of Stem Cells 2023;16:269-80 https://doi.org/10.15283/ijsc23026

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