Figure 3. Clusters/spheroids are composed of internally located ICCs and peripherally located SMCs.

(A) Co-staining with anti-c-Kit and anti-αSMA antibodies. White arrowheads indicate co-expression of c-Kit and αSMA at day 3. Yellow arrowheads indicate cells expressing αSMA but not c-Kit at day 7. A schematic diagram illustrates the spatial arrangement of cells within a day 7 spheroid (green: ICCs; magenta: SMCs). (B) Staining with anti-Desmin antibody, a marker for mature smooth muscle cells. (C) Cell morphology within the spheroid at day 6 visualized by RCAS-gapEGFP expression. (D) Staining of day 5 spheroids with anti-N-cadherin antibody. A white arrowhead indicates N-cadherin-positive cells, while a yellow arrowhead indicates N-cadherin–negative cells in the outer region of the spheroid. Scale bars: 30  µm (A–D), 10  µm (inset a in C).

Figure 3—figure supplement 1. Neuronal markers and TTX response at day 7 organoids.

(A) Co-staining of Day 7 organoids with anti-c-Kit- and Tuj1 antibodies. White arrowhead shows a Tuj1⁺ cell. (B) Representation of Tuj1-positive, αSMA-single positive, c-Kit-single positive cells in an organoid at day 7. Median values: Tuj1⁺, 0.92; αSMA⁺, 40; c-Kit⁺, 53. Sample size: Tuj1⁺, n = 12; αSMA⁺, c-Kit⁺, n=3 (C) Contraction intervals before and after administration of TTX. Median values: Before, 23.4; TTX, 25.35. Sample size: n = 9. Peak counts: Before, 166; TTX, 148. Statistical significance was assessed using Welch’s t-test. n.s., p≥0.05 (p=0.11). Scale bar: 30 µm (A).