Gut contractile organoids: a novel model system to study the cellular synchronization in gastrointestinal motility

概要

Gut contractile organoids: a novel model system
to study the cellular synchronization in gastrointestinal motility
Rei Yagasaki
【Introduction】
Gastrointestinal (GI) motility is important for digestion, absorption, and
excretion. Previous studies have shown that this movement is regulated by smooth muscle
cells (SMCs), enteric nervous system (ENS), and interstitial cells of Cajal (ICCs) known
as a pacemaker. However, how these three types of cells cooperate remains unclear. One
reason is that the GI tract is a multilayered structure hampering analyses at the cellular
level. Therefore, I attempted to analyze GI motility using chicken embryos, which have
a stiff tissue and are easy to manipulate. The biggest problem in the analysis using
chickens has been the absence of available antibodies against the chicken c-Kit protein, a
marker of ICCs. Therefore, I first raised a new antibody against the chicken c-Kit protein,
and verified its usefulness. Next, to analyze GI motility at the cellular level, I developed
a novel in vitro model which is specific to GI motility and then proceeded with the
analysis.
【Materials and Methods】
Anti-c-Kit antibody: The polypeptide at the position of 716–728 amino acids of
the chicken c-Kit protein was synthesized and used for immunization of a rabbit. The
purified antiserum was used as anti-c-Kit antibody. To confirm the specificity of this
antibody against the chicken c-Kit protein, I validated by Western blotting analysis,
immunocytochemistry with c-Kit cDNA-transfected cells, and immunohistochemistry
with c-Kit-expressing tissue.
In vitro model: From an E15 hindgut in chicken embryonic GI tract, by removing
the serosa and intestinal epithelium, only muscle layer was collected. 5.0×105 cells from
muscle layer were cultured with Matrigel. For analysis of this model, I used Ca2+ indicator
GCaMP6s and gap junction inhibitors.
【Results and Discussion】

(1) Anti-c-Kit antibody: Correct signals were detected by this newly raised anti-c-Kit
antibody in Western blotting analysis and immunostaining with c-Kit-overexpressing
cells and tissue. Therefore, I conclude that this new antibody can detect the c-Kit protein
in chickens. Using this antibody, morphology of ICCs in hindgut was also visualized.
ICCs with bipolarity and multipolarity were observed, which is consistent with mammals.
(2) In vitro model: To determine optimal conditions for culturing gut muscle layerderived cells, I tried 3 kinds of FBS-free media on Matrigel. When cultured with
Neurobasal medium, spheroids of cells were formed which exhibited rhythmic
contractions. By immunostaining anti-c-Kit and αSMA antibodies as markers for ICCs
and SMCs, I found that ICCs were located internally whereas SMCs were peripherally.
Based on these characteristics, I designated this spheroid as a gut contractile organoid,
serving as a novel model of GI motility. Visualization of intracellular Ca2+ signals by
GCaMP6s revealed that the organoids exhibited Ca2+ oscillations with the same rhythm
as the contraction. Ca2+ signals were synchronized among organoid-constituting cells.
This synchronization was unaffected by gap junction inhibitors, indicating that there is a
different mechanism other than gap junction-mediated signaling.
Interestingly, when multiple organoids with independent contraction rhythm
fused to form a larger organoid, the rhythm became synchronized in fusing organoids.
These findings revealed interactions between cells and organoids for coordinated
contractions. To further investigate the mechanism underlying the synchronization
between multiple organoids, 3-well hydrogels were prepared that prevented organoidal
fusions. I have found that the rhythm of contraction and Ca2+ oscillations of separated
organoids became synchronized when SMCs bridged separated organoids. The
synchronization between organoids observed in this study recapitulates the local
contractions of the GI motility along the circumferential axis of the gut.
【Conclusion】
I established the gut contractile organoid as a novel model of GI motility. The
organoid serves as a powerful tool for investigating GI motility at the cellular revel. ...