Fig. 3. Reciprocal interaction between colonic organoid and NCSC-derived peripheral neurons. (A) Schematic representation of co-culture of colon organoids with neural crest stem cells (NCSCs). (B) Representative images of NCSCs showing neuronal differentiation after 7 days of co-culture with colon organoids. (C) Representative FACS plot for NCSCs expressing PHOX2B after co-culture with colonic organoids. (D) Increased expression of PHOX2B and NDRG4 in FACS-isolated NCSCs after co-culture with colon organoids. (E) Enrichment of mRNA expression for LMX1A and CHGA in colon organoids after co-culture with NCSCs. (F) Schematic representation of transwell co-culture system. (G) Percentage of PHOX2B-expressing NCSCs induced by co-culture with colon organoids using direct contact or transwells. (H) Verification of PHOX2B-expressing cells after co-culture with colon organoids. (I) Expression of NDRG4 and LMX1A in colon organoids after co-culture with NCSCs using transwells. (J) Differentiated neuronal cells expressing TUJ1 containing serotonin (lower panel) and epithelial cells of colon organoids labeled with eGFP. Columnar morphology of epithelial cells are indicated by dotted red line in the right panel. (K) Proportion of EpCAM-expressing epithelial cells in colon organoids after co-culture with NCSCs. (L) Representative immunostaining image of Ki-67-expressing proliferating cells within colon organoids after co-culture with NCSCs, and the counting result. (M) Representative FACS plot for the isolation of EpCAM-expressing cells of colonic organoid, and representative images for attached cells after re-plating. (N) SOX9 expression in proliferating cells from EpCAM-expressing cells after 10 days of re-cultivation. Data expressed as mean±SD (n=9, 12 for G, biological repeat). p-values calculated by unpaired t-test (D, E, H, I, K, L) or one-way ANOVA (G). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. All scale bars=50 μm. CO: colonic organoid.

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

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