Culture of muscle layer-derived cells prepared from embryonic hindgut.

(A) Culture of muscle layer-derived cells prepared from embryonic hindgut with FBS free-media and substrates. (B) Long-term time-lapse imaging after seeding on Matrigel with Neurobasal media. The images show the ability of these cells to self-assemble at 0, 20, 40, 60, 80 and 100 hours taken from Movie 1. Scale bars: 100 µm (A, B).

Spheroids formed in Neurobasal medium and Matrigel exhibited reiterated contractions.

(A) Clusters/spheroids at days 3, 5, and 7 exhibited reiterative contractions. Graphs show normalized contraction intensities visualized using the Time Measurement function. Arrowheads indicate contraction peaks defined by a peak prominence > 0.25 and a peak width ≤ 10 seconds. (B) Contraction intervals in clusters/spheroids from day 3 to day 7. Each dot represents a single contraction interval. Median values: day 3, 13.3; day 5, 15.4; day 7, 19.6. Sample sizes: day 3, n = 35, peak count = 646; day 5, n = 48, peak count = 748; day 7, n = 21, peak count = 250. Scale bar: 50 µm (A).

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).

Ca2+ imaging of the gut contractile organoid revealed intercellular synchronization.

(A) RCAS-GCaMP6s-P2A-mRuby3 plasmid. (B) Ca2+ imaging of gut contractile organoid during relaxation and contraction. Ca2+ dynamics (green) and normalized values of contraction (grey) of gut contractile organoid. (C) Simultaneous measurement of intercellular Ca2+ dynamics between ICC-ICC, SMC-SMC or ICC-SMC. Three or two ROIs in Ca2+ signal-positive cells were set in a single organoid. Graphs show Ca2+ dynamics in the ROIs. Magnified view shows that a peak of Ca2+ signal in ICC (green) preceded that in SMC (magenta). (D) Peak latency in ICC/SMC. Median value = 0.7; Sample size: n = 14, peak count = 117. Scale bar: 50 µm (B).

Gap junction inhibitor exerted limited effects on the synchronization of Ca2+ dynamics.

Ca²⁺ synchronization among two or three ROIs in GCaMP6s-expressing organoids was evaluated before and after treatment with 100 µM CBX. The synchronization was unaffected between (A) SMC-SMC and (B) ICC-ICC, but was partially affected between (C) ICC–SMC. Magnified views (a, b) highlight that the preceding Ca²⁺ peak in ICC (green) observed before CBX treatment (a) was abolished after treatment (b). (D) Peak latency between ICC and SMC. Median values: Before, 1.167; CBX 20 µM, 0.701; CBX 100 µM, 0.0. Statistical significance was assessed using Welch’s t-test. ***p < 0.001, n.s., p ≥ 0.05 (p = 0.27 for Before vs. CBX 20 µM). Sample sizes: Before: n = 10, peak count = 50; CBX 20 µM: n = 5, peak count = 34; 100 µM: n = 5, peak count = 51, Scale bar: 50 µm (A).

The organoidal contraction is important for Ca2+ dynamics in ICCs.

GCaMP6s-expressing organoids were cultured with Blebbistatin. (A) Organoidal contractions were extinguished at 10 µM. (B) Contraction intervals before and after Blebbistatin, and upon washout. Median values: Before, 33.46; Blebbistatin 5 µM, 12.86; 10 µM, N/A; Washout, 23.95. Statistical significance was assessed using Welch’s t-test. ***p < 0.001, *p < 0.05. Sample size: n = 3. (C-E) Comparison of Ca2+ dynamics in SMC-SMC, ICC-ICC, and ICC-SMC. Three or two ROIs were assessed before and after administrations of 10 µM Blebbistatin. (F) Ca2+ transients in a single ICC at 0 μM (before), 5 μM, and 10 μM. Scale bar: 50 µm (B).

Nifedipine ceased organoidal contractions and Ca2+ activities in ICCs.

Similar experiment to Fig. 6. (A) Organoidal contractions were extinguished at 1 µM. (B) Contraction intervals before and after administration, and upon washout. Median values: Before, 25.7; Nifedipine 1 µM, N/A; Washout, 22.2. Statistical significance was assessed using Welch’s t-test. **p < 0.01. Sample size: n = 4. Peak counts: Before, 101; Nifedipine 1 µM, N/A; Washout, 46. (C) Ca²⁺ transients in a single ICC before and after administration of 1 µM nifedipine, and following washout. Scale bar: 50 µm (B).

Ca2+ transients in multiple organoids undergo synchronization upon organoidal fusion.

(A) Time-lapse imaging of organoidal fusion. (B) When two organoids that originally displayed independent Ca2+ rhythm fused to each other, their rhythm became synchronized after fusion (24 h). (C) Contraction intervals before and after fusion. Median values: before fusion, 13.65; after fusion, 21.04. Statistical significance was assessed using Welch’s t-test. ***p < 0.001. Sample size: n = 3 pairs. Peak counts: before, 138; after, 90. (D) Cellular protrusions between two neighboring organoids. White arrowheads show three protrusions from the left organoid. Scale bars: 100 µm (A, B, D), 20 µm (inset (a) in (D)).

Smooth muscle cells mediate Ca2+ synchronization between organoids.

(A) Diagram of a 3-well hydrogel in which one organoid was placed per well. The three wells were connected with narrow channels, and this gel mold does not allow organoidal bodies to fuse to each other, but allow them to extend/migrate protrusions/cells through the channel (B). (C) After 3 days, the three organoids displayed synchronization of Ca2+ dynamics. (D) Some organoid-derived cells crawled out from the wells, and covered the top surface of the hydrogel, resulting in bridging the three unfused organoids. (E) Diagram of a 3-well hydrogel without channels. (F) The surface-covering cells were identified as SMCs (αSMA-positive, c-Kit-negative). (G) In the hydrogel without channels but with surface-covered SMCs, Ca2+ dynamics in the three organoids were synchronized. (H) The Ca²⁺ synchronization shown in (G) was not altered by treatment with 18β-GA. Blue planes indicate focal planes. Scale bars: 50 µm (B); 100 µm (C, D, F, G, H).

(A) Chicken embryonic hindgut at E15. It was dissected from the bottom of the cecum to the front of the cloaca (white lines). (B) Three layers of E15 hindgut: serosa, muscle layer, intestinal epithelium. Remak’s ganglion was also removed. Scale bars: 1 mm in A, B

(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).

Effects of gap junction inhibitors on organoidal contractions and ICC-SMC latency.

(A) Organoids at day 7 exhibited rhythmic contractions before and after administration of 100 µM CBX. Graphs show normalized contraction intensities visualized using the Time Measurement function. Arrowheads indicate contraction peaks defined by a peak prominence > 0.25 and a peak width ≤ 10 seconds. (B) Contraction intervals before and after treatment with CBX (100 µM), H₂O (vehicle control for CBX), 18β-GA (100 µM), and DMSO (vehicle control for 18β-GA). Median values: CBX, 14.0 (before), 11.9 (after); H₂O, 14.7 (before), 15.4 (after); 18β-GA, 14.7 (before), 7.7 (after); DMSO, 19.95 (before), 17.5 (after). Sample sizes and peak counts: CBX, n = 9 (before), n = 9 (after), peak count = 150 (before), 196 (after); H₂O, n = 10 (before), n = 10 (after), peak count = 173 (before), 171 (after); 18β-GA, n = 7 (before), n = 8 (after), peak count = 129; DMSO, n = 7 (before), n = 6 (after), peak count = 193 (before), 92 (after). (C) Contraction intervals at day 7 in organoids cultured with CBX, H₂O, 18β-GA, and DMSO from day 3 to day 7. Median values: H₂O, 22.75; CBX, 32.5; DMSO, 18.85; 18β-GA, 27.95. Sample sizes and peak counts: H₂O, n = 9, peak count = 136; CBX, n = 11, peak count = 135; DMSO, n = 9, peak count = 209; 18β-GA, n = 18, peak count = 268. (D) Peak latency of Ca2+ transients between ICC and SMC before and after administration of 18β-GA (20 µM, 100 µM). Statistical significance was assessed using Welch’s t-test. n.s., p = 0.27 for Before vs. 18β-GA 20 µM; p = 0.25 for Before vs. 18β-GA 100 µM; p = 0.46 for 18β-GA 20 µM vs. 100 µM. Sample sizes and peak counts: Before, n = 5, peak count = 62; 18β-GA 20 µM, n = 3, peak count = 52; 18β-GA 100 µM, n = 2, peak count = 57. (E) Contractions of cultured embryonic heart cells. Graphs show normalized contraction intensities using the Time Measurement function. The box plot shows the number of contractions per 5 minutes. Median values: CBX, 30.75 (before), 15(CBX 100µM), 55.75 (washout); 18β-GA: 45.5 (before), 8.5 (18β-GA 100µM), 51 (washout). Statistical significance was assessed using Welch’s t-test. **p < 0.01, *p < 0.05; n.s., p ≥ 0.05 (p = 0.13 for Before vs. washout of CBX; p = 0.26 for Before vs. washout of 18β-GA). Sample sizes: CBX, n = 4; 18β-GA, n = 4. Scale bar: 50 µm (A).