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.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Gene Expression Omnibus
Furness, J.B. The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).
Sasselli, V., Pachnis, V. & Burns, A.J. The enteric nervous system. Dev. Biol. 366, 64–73 (2012).
Obermayr, F., Hotta, R., Enomoto, H. & Young, H.M. Development and developmental disorders of the enteric nervous system. Nat. Rev. Gastroenterol. Hepatol. 10, 43–57 (2013).
Saffrey, M.J. Cellular changes in the enteric nervous system during ageing. Dev. Biol. 382, 344–355 (2013).
McKeown, S.J., Stamp, L., Hao, M.M. & Young, H.M. Hirschsprung disease: a developmental disorder of the enteric nervous system. Wiley Interdiscip. Rev. Dev. Biol. 2, 113–129 (2013).
Burns, A.J. & Thapar, N. Neural stem cell therapies for enteric nervous system disorders. Nat. Rev. Gastroenterol. Hepatol. 11, 317–328 (2014).
Hao, M.M. & Young, H.M. Development of enteric neuron diversity. J. Cell. Mol. Med. 13, 1193–1210 (2009).
Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).
McCracken, K.W., Howell, J.C., Wells, J.M. & Spence, J.R. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat. Protoc. 6, 1920–1928 (2011).
Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).
Watson, C.L. et al. An in vivo model of human small intestine using pluripotent stem cells. Nat. Med. 20, 1310–1314 (2014).
Bajpai, R. et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463, 958–962 (2010).
Mica, Y., Lee, G., Chambers, S.M., Tomishima, M.J. & Studer, L. Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Rep. 3, 1140–1152 (2013).
Kudoh, T., Wilson, S.W. & Dawid, I.B. Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129, 4335–4346 (2002).
Fu, M., Tam, P.K., Sham, M.H. & Lui, V.C. Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study. Anat. Embryol. (Berl.) 208, 33–41 (2004).
Young, H.M., Ciampoli, D., Hsuan, J. & Canty, A.J. Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut. Dev. Dyn. 216, 137–152 (1999).
Young, H.M. et al. GDNF is a chemoattractant for enteric neural cells. Dev. Biol. 229, 503–516 (2001).
Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Huebsch, N. et al. Automated video-based analysis of contractility and calcium flux in human-induced pluripotent stem cell-derived cardiomyocytes cultured over different spatial scales. Tissue Eng. Part C Methods 21, 467–479 (2015).
Hao, M.M. et al. Enteric nervous system assembly: functional integration within the developing gut. Dev. Biol. 417, 168–181 (2016).
Bohórquez, D.V. et al. Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells. J. Clin. Invest. 125, 782–786 (2015).
Bajaj, R. et al. Congenital central hypoventilation syndrome and Hirschsprung's disease in an extremely preterm infant. Pediatrics 115, e737–e738 (2005).
Pattyn, A., Morin, X., Cremer, H., Goridis, C. & Brunet, J.F. The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399, 366–370 (1999).
Fu, M., Lui, V.C., Sham, M.H., Cheung, A.N. & Tam, P.K. HOXB5 expression is spatially and temporarily regulated in human embryonic gut during neural crest cell colonization and differentiation of enteric neuroblasts. Dev. Dyn. 228, 1–10 (2003).
Lui, V.C. et al. Perturbation of hoxb5 signaling in vagal neural crests down-regulates ret leading to intestinal hypoganglionosis in mice. Gastroenterology 134, 1104–1115 (2008).
Denham, M. et al. Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system. Stem Cells 33, 1759–1770 (2015).
Wallace, A.S. & Burns, A.J. Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract. Cell Tissue Res. 319, 367–382 (2005).
Kabouridis, P.S. et al. Microbiota controls the homeostasis of glial cells in the gut lamina propria. Neuron 85, 289–295 (2015).
Bergner, A.J. et al. Birthdating of myenteric neuron subtypes in the small intestine of the mouse. J. Comp. Neurol. 522, 514–527 (2014).
Erickson, C.S. et al. Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines. Neurogastroenterol. Motil. 26, 874–884 (2014).
Baetge, G. & Gershon, M.D. Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons. Dev. Biol. 132, 189–211 (1989).
Blaugrund, E. et al. Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence. Development 122, 309–320 (1996).
Anlauf, M., Schäfer, M.K., Eiden, L. & Weihe, E. Chemical coding of the human gastrointestinal nervous system: cholinergic, VIPergic, and catecholaminergic phenotypes. J. Comp. Neurol. 459, 90–111 (2003).
Anderson, G. et al. Loss of enteric dopaminergic neurons and associated changes in colon motility in an MPTP mouse model of Parkinson's disease. Exp. Neurol. 207, 4–12 (2007).
Burns, A.J. et al. White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies. Dev. Biol. 417, 229–251 (2016).
Fattahi, F. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105–109 (2016).
Hotta, R. et al. Transplanted progenitors generate functional enteric neurons in the postnatal colon. J. Clin. Invest. 123, 1182–1191 (2013).
Lindley, R.M. et al. Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon. Gastroenterology 135, 205–216 (2008).
Burns, A.J., Roberts, R.R., Bornstein, J.C. & Young, H.M. Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages. Semin. Pediatr. Surg. 18, 196–205 (2009).
Miyaoka, Y. et al. Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat. Methods 11, 291–293 (2014).
Costa, M. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792–796 (2007).
Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).
Tang, W. et al. Faithful expression of multiple proteins via 2A-peptide self-processing: a versatile and reliable method for manipulating brain circuits. J. Neurosci. 29, 8621–8629 (2009).
Lee, G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468–1475 (2007).
Chen, J., Bardes, E.E., Aronow, B.J. & Jegga, A.G. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 37, W305–11 (2009).
Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6, e21800 (2011).
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.
The authors declare no competing financial interests.
Supplementary Figures 1–10 & Supplementary Tables 1–2 (PDF 1203 kb)
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)
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)
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)
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)
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)
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)
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)
About this article
Cite this article
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
This article is cited by
Journal of Hematology & Oncology (2023)
Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility
Veterinary Research (2023)
Nature Reviews Genetics (2023)
Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche
Nature Communications (2023)
Surgery Today (2023)