Developmental Biology

The gut contractile organoid: a novel model for studying the gut motility regulated by coordinating signals between interstitial cells of Cajal and smooth muscles

Rei Yagasaki
Ryo Nakamura
Yuuki Shikaya
Ryosuke Tadokoro
Ruolin Hao
Zhe Wang
Mototsugu Eiraku
Masafumi Inaba
Yoshiko Takahashi author has email address

Department of Zoology, Graduate School of Science, Kyoto University Sakyo-ku, Kyoto 606-8502, Japan
Institute for Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

https://doi.org/10.7554/eLife.97860.1

Open access
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Figures and data

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 in A, B.

Forming spheroids exhibited reiterated contractions.
(A) Clusters/spheroids on day 3, 5 and 7 exhibited periodic contractions. Graphs show contractions quantified by differences in the intensity of brightfield in sequential images. (B) Frequency of contractions of the cluster/spheroid on day 3-7 (N=day 3: 46, day 5: 35, day 7: 60). The number on each graph shows the average of contraction frequencies. Scale bars: 50 µm in A.

The cluster/spheroid is composed of ICCs internally and SMCs peripherally.
(A) Co-staining with anti-c-Kit- and αSMA antibodies. White arrowheads show co-expression of c-Kit and αSMA at Day 3. Yellow arrowheads indicate a cell expressing αSMA but not c-Kit at day 7. A diagram shows an image of cell arrangement (green: ICC, magenta: SMC) in the spheroid at day 7. (B) Cell morphology in the spheroid at day 6 revealed by RCAS-gapEGFP expression. (C) Staining of spheroids at day 5 with anti-N-cadherin antibody. A white arrowhead shows expression of N-cadherin. A yellow arrowhead shows a cell which does not express N-cadherin in the outer region of the spheroid. Scale bars: 30 µm in A-C, 10 µm in a.

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 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 with Ca²⁺ signal-positive cells were set in a single organoid. Graphs show Ca²⁺ dynamics in the ROIs. Magnified view shows that a rise of Ca²⁺ signal in ICC (green) preceded that in SMC (magenta). Scale bar: 50 µm in B.

Gap junction inhibitor exerted limited effects on the synchronization of Ca²⁺ dynamics.
GCaMP6s-expressing organoids were cultured with CBX, and three or two ROIs were assessed for their Ca²⁺ synchronization. A single organoid was subjected to the assessment before (0 µM; control) and after administrations of 100 µM CBX. The synchronization was unaffected between (A) SMC-SMC and (B) ICC-ICC, but that in (C) ICC-SMC was partially affected. Magnified view shows that the preceding rise of Ca²⁺ in ICC (green) before CBX was abrogated after CBX. Scale bar: 50 µm in A.

The organoidal contraction is important for Ca²⁺ dynamics in ICCs.
GCaMP6s-expressing organoids were cultured with Blebbistatin. (A) Ca²⁺ oscillation was extinguished in the entire organoid by administration of Blebbistatin. (B-D) Ca²⁺ dynamics in (B) SMC-SMC, (C) ICC-ICC and (D) ICC-SMC. Three or two ROIs were assessed for their Ca²⁺ synchronization. A single organoid was subjected to the assessment before (0 µM; control) and after administrations of 10 µM Blebbistatin. (E) Ca²⁺ transients in a single ICC in 0 μM, 5 μM, and 10 μM. Scale bar: 50 µm in B.

Ca²⁺ dynamics in multiple gut contractile organoids undergo synchronization upon their fusion.
(A) Time-lapse imaging of organoidal fusion. (B) When two organoids that originally showed independent rhythm of Ca²⁺ fused to each other, their rhythm became synchronized after fusion (24 h). (C) Protrusions between two neighboring organoids. White arrowheads show three protrusions from the left organoid. Scale bars: 100 µm in A-C, 20 µm in a.

Smooth muscle cells mediate the Ca²⁺ synchronization between organoids.
(A) Diagram of 3-well hydrogel in which an organoid placed in each well. This gel mold does not allow organoidal bodies to fuse to each other, but allow them to extend/migrate protrusions/cells through a narrow channel connecting the wells (B). (C) After 3 days, 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 3-well hydrogel without channels (F) Surface-covering cells were SMCs (αSMA positive and c-Kit-negative). (G) In the hydrogel without channels but with surface-covered SMCs, Ca²⁺ dynamics in the 3 organoids were synchronized. (H) The Ca²⁺ synchronization shown in (G) was not altered by 18β-GA. Scale bars: 50 µm in B, 100 µm in C-H.

(A) Chicken embryonic hindgut at E15. The hindgut 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⁺ cells in an organoid at day 7. Scale bar: 30 µm in A.

(A) Day 7 organoid exhibited periodic contractions both before (0 µM; control) and after adding CBX. Graphs show contractions quantified by the difference in intensity of brightfield in sequential images. (B) Relative changes to the control in contraction frequency in day 7 organoid after CBX. The number is an average. Scale bar: 50 µm in A.

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