Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Furness, J.B. The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Sasselli, V., Pachnis, V. & Burns, A.J. The enteric nervous system. Dev. Biol. 366, 64–73 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. 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).

    Article  CAS  PubMed  Google Scholar 

  4. Saffrey, M.J. Cellular changes in the enteric nervous system during ageing. Dev. Biol. 382, 344–355 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. 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).

    Article  CAS  PubMed  Google Scholar 

  6. Burns, A.J. & Thapar, N. Neural stem cell therapies for enteric nervous system disorders. Nat. Rev. Gastroenterol. Hepatol. 11, 317–328 (2014).

    Article  PubMed  Google Scholar 

  7. Hao, M.M. & Young, H.M. Development of enteric neuron diversity. J. Cell. Mol. Med. 13, 1193–1210 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

    Article  PubMed  CAS  Google Scholar 

  9. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  PubMed  CAS  Google Scholar 

  11. Watson, C.L. et al. An in vivo model of human small intestine using pluripotent stem cells. Nat. Med. 20, 1310–1314 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bajpai, R. et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463, 958–962 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  PubMed  Google Scholar 

  17. Young, H.M. et al. GDNF is a chemoattractant for enteric neural cells. Dev. Biol. 229, 503–516 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hao, M.M. et al. Enteric nervous system assembly: functional integration within the developing gut. Dev. Biol. 417, 168–181 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Bohórquez, D.V. et al. Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells. J. Clin. Invest. 125, 782–786 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Bajaj, R. et al. Congenital central hypoventilation syndrome and Hirschsprung's disease in an extremely preterm infant. Pediatrics 115, e737–e738 (2005).

    Article  PubMed  Google Scholar 

  23. 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).

    Article  CAS  PubMed  Google Scholar 

  24. 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).

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  CAS  PubMed  Google Scholar 

  26. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    Article  PubMed  Google Scholar 

  28. Kabouridis, P.S. et al. Microbiota controls the homeostasis of glial cells in the gut lamina propria. Neuron 85, 289–295 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bergner, A.J. et al. Birthdating of myenteric neuron subtypes in the small intestine of the mouse. J. Comp. Neurol. 522, 514–527 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    Article  CAS  PubMed  Google Scholar 

  32. 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).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fattahi, F. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105–109 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hotta, R. et al. Transplanted progenitors generate functional enteric neurons in the postnatal colon. J. Clin. Invest. 123, 1182–1191 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 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).

    Article  PubMed  Google Scholar 

  39. 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).

    Article  PubMed  Google Scholar 

  40. Miyaoka, Y. et al. Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat. Methods 11, 291–293 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Costa, M. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792–796 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Article  CAS  PubMed  Google Scholar 

  45. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6, e21800 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4233

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing