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Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system

Abstract

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

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Figure 1: Incorporation of NCCs into developing HIOs in vitro.
Figure 2: Formation of a three-dimensional neuronal plexus in HIOs+ENS grown in vivo.
Figure 3: Live imaging of neural activity in HIOs+ENS.
Figure 4: ENS-independent and dependent control of contractile activity.
Figure 5: ENS effects on the epithelium.
Figure 6: Modeling a Hirschsprung's-disease-causing mutation in PHOX2B.

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Acknowledgements

We thank A. Zorn, N. Shroyer and members of the Wells and Zorn laboratories for reagents and feedback. We thank M. Kofron for assistance with confocal imaging. We thank S. Danzer, R. Pun, J. Piero, M. Marotta and M. Oria for help with the equipment for the electrical field stimulation experiments. We thank K. Campbell and J. Kuerbitz for providing antibodies for the neurochemical analysis. This work was supported by US National Institutes of Health grants U18TR000546 (J.M.W.), U18EB021780 (J.M.W. and M.A.H.), U01DK103117 (J.M.W. and M.A.H.), R01DK098350 (J.M.W.) and R01DK092456 (J.M.W.), and an Athena Blackburn Research Scholar Award in Neuroenteric Diseases (M.M.M.). We also acknowledge core support from the Cincinnati Digestive Disease Center Award (P30 DK0789392; Pilot and Feasibility Award), Clinical Translational Science Award (U54 RR025216) and technical support from Cincinnati Children's Hospital Medical Center (CCHMC) Confocal Imaging Core and the CCHMC human Pluripotent Stem Cell Facility.

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Authors

Contributions

M.J.W., M.M.M. and J.M.W. conceived the study and experimental design, performed and analyzed experiments and wrote the manuscript. M.M.M., H.M.P., C.L.W., N.S. and M.A.H. helped to design and execute the mouse engraftment experiments, and M.M.M. performed the functional ENS assays. S.T. performed the experiments using the PHOX2B lines. P.A. and M.N. helped to design and execute the ex vivo organ-bath studies. S.A.B and C.-F.C. designed and performed the chick experiments. B.R.C., M.A.M. and Y.M. suggested the use of and provided the GCaMP6f and PHOX2B induced PSC lines. A.G.E., J.S. and E.G.S. provided the GAPDH-GFP HESC line. All of the authors contributed to the writing or editing of the manuscript.

Corresponding authors

Correspondence to Michael A Helmrath or James M Wells.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–10 & Supplementary Tables 1–2 (PDF 1203 kb)

3-dimensional image of human intestine showing enteric nerves in association with smooth muscle.

Nerves were stained with TUBB3 (green) and smooth muscle was stained with Desmin (red). Nerves were tightly integrated into the layers of smooth muscle. Video corresponds to Supplementary Fig. 7a, top left panel. (MP4 19701 kb)

3-dimensional image of HIOs+ENS tissue grown in vivo showing human enteric nerves in association with smooth muscle.

Nerves were stained with TUBB3 (green) and smooth muscle was stained with Desmin (red). NCC-derived nerves were embedded within the layers of smooth muscle both in the myenteric and submucosal layers. Video corresponds to Supplementary Fig. 7a, top right panel. (MP4 9473 kb)

Time-lapse video of HIOs+ENS in vitro where the ENS was derived from neural crest cells containing a GCaMP6f reporter

Twenty-minute time-lapse video of HIOs+ENSshowing Ca2+ flux specifically in NCC-derived cells. HIOswere generated with H1 cells, which do not have a Ca2+indicator. Single neurons have regular periodicity of depolarization. Video corresponds to Fig. 3a. (MP4 12045 kb)

KCl stimulation of HIOs+ENS in vitro.

Time-lapse video of HIOs+ENS showing broad depolarization of NCC-derived ENS cells in response to KCl addition. NCCs were generated from GCaMP6f expressing iPSCs. Video corresponds to Fig. 3b. (MP4 23241 kb)

Time-lapse video of explanted HIOs+ENS derived in vivo using GCaMP6f neural crest cells

A large nerve fiber was imaged where calcium oscillation was observed. NCCs were generated from GCaMP6f expressing iPSCs. Video corresponds to Fig. 3c, left panel. (MP4 2008 kb)

KCl stimulation of explanted HIOs+ENS derived in vivo using GCaMP6f neural crest cells.

Time-lapse video of transplanted HIOs+ENS showing depolarization of NCCderived ENS cells in response to KCl addition. NCCs were generated from GCaMP6f expressing iPSCs. Video corresponds to Fig. 3c, right panel. (MP4 7784 kb)

Time-lapse videos of electrically stimulated HIOs grown in vivo.

Video 7 corresponds to the left panel of Fig. 4a (HIO) and shows an HIO lacking enteric nerves. Video 8 corresponds to the middle panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells. Video 9 corresponds to the right panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells that were stimulated in the presence of tetrodotoxin (HIO+ENS + TTX). Videos are played at 16X play speed. (MP4 2143 kb)

Time-lapse videos of electrically stimulated HIOs grown in vivo.

Video 7 corresponds to the left panel of Fig. 4a (HIO) and shows an HIO lacking enteric nerves. Video 8 corresponds to the middle panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells. Video 9 corresponds to the right panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells that were stimulated in the presence of tetrodotoxin (HIO+ENS + TTX). Videos are played at 16X play speed. (MP4 2891 kb)

Time-lapse videos of electrically stimulated HIOs grown in vivo.

Video 7 corresponds to the left panel of Fig. 4a (HIO) and shows an HIO lacking enteric nerves. Video 8 corresponds to the middle panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells. Video 9 corresponds to the right panel of Fig. 4a (HIO+ENS) and shows an HIO containing engrafted neural crest cells that were stimulated in the presence of tetrodotoxin (HIO+ENS + TTX). Videos are played at 16X play speed. (MP4 3948 kb)

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Workman, M., Mahe, M., Trisno, S. et al. Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat Med 23, 49–59 (2017). https://doi.org/10.1038/nm.4233

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