The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord1. The ENS has been called the ‘second brain’1 given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon2. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrbs-l/s-l), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.
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We thank K. Manova, M. Tomishima and A. Viale for technical assistance and G. Lee for human ES-cell-based PHOX2B–GFP reporter line. We are grateful for technical support provided by H. S. Ralph, K. Brodie and the MSKCC Flow Cytometry Core. We would also like to thank J.-F. Brunet for providing us with the PHOX2A antibody and P. Frykman for sharing their EDNRBtm1Ywa/FrykJ HSCR mouse strain. The work was supported by the Starr Foundation and by NYSTEM contract C026446 to L.S. and by P30 CA008748; by grant NS15547 from the NINDS to M.D.G. and by grants RN200946-1 and RN3-06425 from the California Institute for Regenerative Medicine (CIRM) to T.C.G; by TRI-SCI 2014-030 to L.S. and S.C., and by the New York Stem Cell Foundation (R-103) and NIDDK (DP2 DK098093-01) to S.C. S.C. is a New York Stem Cell Foundation – Robertson Investigator. J.A.S. was supported by a DFG fellowship. We would like to thank M. Tomishima and V. Tabar for comments on the manuscript.
The authors declare no competing financial interests.
Extended data figures and tables
a, Schematic illustration of CNC and MNC induction protocols3. b, Flow cytometry for CD49D and SOX10–GFP in CNC and MNC cells. c, Immunofluorescence of unsorted and CD49D-sorted differentiated NC cells for SOX10. d, Flow cytometry for CD49D in ENCs derived from H9 human ES cells and control and familial dysautonomia (FD) human iPS cells. e, f, Representative immunofluorescence images and flow cytometry in ES-cell-derived ENC for enteric precursor lineage marker at day 11. g, List of the top 10 and selected additional (bold) most differentially expressed transcripts from the RNA-seq analysis of CNC compared to stage matched CNS precursors31. h, Lists of the top 10 and selected additional (bold) most differentially expressed transcripts from the RNA-seq analysis of MNC compared to stage-matched CNS precursors31. i, Distribution of CNC and MNC cells in developing chick embryos at 24–36 h after injection. Right, higher power image of the clusters of MNC cells in the developing surface ectoderm. NotoC, notochord; NT, neural tube; S, somite. Scale bars, 50 μm (c, i, middle), 25 μm (e, i, right) and 1 mm (i, left).
a, Flow cytometry for TRKC and PHOX2A expression. b, Immunofluorescence for PHOX2A and ASCL1 for TRKC-positive and TRKC-negative ES-cell-derived ENC precursors. c, Time course qRT–PCR analysis of enteric lineage markers during more extended in vitro differentiation periods; n = 3 independent experiments. d, e, Flow cytometric quantification of enteric neuron precursor and neuronal markers in ENC-derived neurons at days 40 and 60 of differentiation; n = 3 independent experiments. Scale bars, 50 μm.
a–c, Representative immunofluorescence images for expression of 5-HT, TUJ1 and tyrosine hydroxylase (TH) in CNC-derived neurons. In contrast to ENC-derived lineages, no serotonergic (5-HT+) neurons were detected under cranial conditions despite the presence of many TUJ1+ neurons and increased percentages of tyrosine-hydroxylase-positive cells. d, e, Flow cytometry for TRKB and TRKC under CNC and ENC conditions; n = 3 independent experiments. Scale bars, 50 μm.
Extended Data Figure 4 Functional characterization of ES-cell-derived enteric neurons in co-culture with SMCs.
a, Schematic illustration of SMC differentiation protocol. b, Immunofluorescence staining of SMC progenitors for SMA and ISL1. c, Association of various ENC-derived neuron subtypes with SMA+ cells. d, SYN–eGFP expression in ENC-derived neurons at 40 days of co-culture with SMCs and stage-matched neurons in the absence of SMCs. e, Monoculture of SMCs versus co-cultures of SMCs with ENC-derived neurons. Top, phase-contrast images showing morphological changes of SMCs in co-culture. Bottom, immunofluorescence staining of mature SMC markers MYH11 and AchR in monoculture of SMCs versus co-cultures of SMCs with ENC-derived neurons. f, Diagrams representing extent of contraction of SMC cultures. Arrows indicate the time of pharmacological stimulation. Scale bars, 50 μm (b, c, e) and 100 μm (d).
Extended Data Figure 5 Generation of tissue-engineered colon using human ES-cell-derived ENC precursors.
a, Schematic illustration for generation of tissue-engineered colon. b, Tissue engineered colon stained for human cytoplasmic marker SC121, TUJ1 and human-specific synaptophysin (hSyn). Dotted line shows approximate location of border between muscle and epithelial/submucosal-like layers. H&E, haematoxylin and eosin. Scale bars, 20 μm (b, left and middle) and 40 μm (b, right).
Extended Data Figure 6 Characterization of transplanted human ES-cell-derived ENC precursors in adult colon of NSG and Ednrbs-l/s-l mice.
a, Whole-mount microscopy of colon transplanted with RFP+ CD49D-purified ES-cell-derived ENC precursors to track RFP expression at injection site, at 1 h after transplantation to ensure that cells were injected at proper location (left), and at 2 weeks to show dispersal of the cells and congregation of subset of cells into distinct clusters (right). The dashed lines indicate the outer border of the intact colon tissue. b, c, Whole-mount fluorescence imaging and quantification of migration of grafted RFP+ ES-cell-derived CNS precursors and CD49D-purified CNC precursors inside the adult colon wall. d, Megacolon-like phenotype in control animals versus animals receiving ES-cell-derived ENC transplants. e, f, Whole-mount fluorescence imaging and quantification of migration of grafted RFP+ CD49D purified ES-cell-derived ENC precursors in colon of Ednrbs-l/s-l mice. g, Total gastrointestinal transit times by carmine dye gavage of Ednrbs-l//s-l mice grafted with RFP+ CD49D-purified ES-cell-derived ENC precursors versus Matrigel-only grafted mice; n = 3 for grafted animals, n = 2 for Matrigel group. Note that n = 2 for Matrigel group was due to the fact that nearly all Matrigel-injected animals died owing to their disease phenotype. h, Representative images of grafted ES-cell-derived ENC precursors at 3 months after transplantation into the colon of Ednrbs-l/s-l mice co-expressing TUJ1 and SC121. i, Immunofluorescence staining of cross sections of Ednrbs-l/s-l colons transplanted with Matrigel for SC121 and human-specific synaptophysin. j, Representative image of grafted ES-cell-derived ENC precursors at 3 months after transplantation into the colon of Ednrbs-l/s-l mice expressing human-specific GFAP (SC123). k, l, Representative images of grafted ES-cell-derived ENC precursors at 6 weeks after transplantation into the colon of NSG (wild type) and Ednrbs-l/s-l mice. The dashed lines indicate the border between submucosal and muscle layers. Scale bars, 200 μm (a), 1 cm (b, e) and 100 μm (h–l). AU, arbitrary units. P value for g is given numerically, t-test with Welch’s correction; n = 3 independent experiments.
a, Sequences of wild-type and Cas9-nickase induced bi-allelic nonsense mutations in targeted region of Ednrb−/− clones. b, Western blot analysis for EDNRB in ES-cell-derived ENC precursors showing lack of protein expression in the mutant clones C1–C4. c, Ednrb−/− human ES cells can be efficiently differentiated into CD49D+ human ES-cell-derived ENC based on CD49D expression (c) and expression of SOX10 (data not shown). d, Proliferation of Ednrb−/− human ES-cell-derived ENCs (day 11) versus wild-type-derived cells; n = 4 independent experiments. e, LDH activity measurement of cell viability in Ednrb−/− ES-cell-derived ENCs (day 11) versus wild-type-derived cells. *P < 0.05 (t-test; n = 3 independent experiments).
Extended Data Figure 8 Chemical screening for compounds that rescue migration of Ednrb−/− ES-cell-derived NC precursors.
a, Schematic illustration of the timeline and experimental steps involved in the chemical screening assay and migration scoring system. b, Example of a screening plate layout and locations of dimethylsulfoxide (DMSO) control wells. c, Migration scores of Prestwick library compounds and DMSO controls. d, Migration assay and scores for Ednrb−/− ES-cell-derived ENC precursors treated with primary hit compounds. Z-score for primary hit compounds in c is given numerically (compared to DMSO control; n = 224 technical replicates).
Extended Data Figure 9 Pharmacological modulation of migration in human ES-cell-derived ENC precursors.
a, BACE2 expression in the various human ES-cell-derived NC sublineages at day 11 as compared to stage-matched CNS precursors. b, qRT–PCR analysis to confirm knockdown of BACE2 in CD49D-purified Ednrb−/− ES-cell-derived ENCs after siRNA transfection compared to control siRNA. c, Quantification of whole-mount images of the colon of NSG mice transplanted with RFP+ CD49D-purified wild-type and Ednrb−/− ES-cell-derived ENC precursors, with or without pepstatin A pre-treatment (compare to Fig. 4j). d, Representative images of wild-type CD49D-purified ES-cell-derived ENCs treated with EDN3 and BQ-788 (EDNRB inhibitor). e, Colon migration assay in wild-type ES-cell-derived ENC precursors after pre-treatment with BQ-788. f, Quantification of the data in e. AU, arbitrary unit. Scale bar, 1 cm.
This file contains Supplementary Tables 1-4. Supplementary Table 1 contains the top 200 most differentially expressed transcripts in ENC (vagal/enteric NC) versus CNS lineage cells from RNA seq data; Supplementary Table 2 contains the top 200 most differentially expressed transcripts in CNC (cranial NC) versus CNS lineage cell from RNA seq data; Supplementary Table 3 contains the top 200 most differentially expressed transcripts in MNC (melanocyte-biased) versus CNS lineage cells from RNA seq data and Supplementary Table 4 contains a list of primary antibodies and working dilutions. (ZIP 49 kb)
Contractile response of co-culture of SMCs with ENC-derived neurons following exposure to Acetylcholine.
Contractile response of co-culture of SMCs with ENC-derived neurons following exposure to Acetylcholine. (MP4 9152 kb)
Contractile response of co-culture of SMCs with ENC-derived neurons following exposure to Carbachol.
Contractile response of co-culture of SMCs with ENC-derived neurons following exposure to Carbachol. (MP4 9251 kb)
Contractile response of co-culture of SMCs with ENC-derived neurons following KCl-mediated depolarization.
Contractile response of co-culture of SMCs with ENC-derived neurons following KCl-mediated depolarization. (MP4 8887 kb)
Contractile response of monoculture of SMCs following exposure to Acetylcholine. (MP4 9100 kb)
Contractile response of monoculture of SMCs following exposure to Carbachol. (MP4 7489 kb)
Contractile response of monoculture of SMCs following KCl-mediated depolarization. (MP4 7170 kb)
Contractile response of SMCs prior to and during optogenetic light stimulation at increasing frequencies of light pulses (450 nm).
Contractile response of SMCs prior to and during optogenetic light stimulation at increasing frequencies of light pulses (450 nm). (MP4 15110 kb)
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Fattahi, F., Steinbeck, J., Kriks, S. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105–109 (2016). https://doi.org/10.1038/nature16951
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