The enteric nervous system (ENS) represents a vast network of neuronal and glial cell types that develops entirely from migratory neural crest (NC) progenitor cells. Considerable improvements in the understanding of the molecular mechanisms underlying NC induction and regional specification have recently led to the development of a robust method to re-create the process in vitro using human pluripotent stem cells (hPSCs). Directing the fate of hPSCs toward the enteric NC (ENC) results in an accessible and scalable in vitro model of ENS development. The application of hPSC-derived enteric neural lineages provides a powerful platform for ENS-related disease modeling and drug discovery. Here we present a detailed protocol for the induction of a regionally specific NC intermediate that occurs over the course of a 15-d interval and is an effective source for the in vitro derivation of functional enteric neurons (ENs) from hPSCs. Additionally, we introduce a new and improved protocol that we have developed to optimize the protocol for future applications in regenerative medicine, in which components of undefined activity have been replaced with fully defined culture conditions. This protocol provides access to a broad range of human ENS lineages within a 30-d period.
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The data generated or analyzed during this study are included in this published article (and its Supplementary Information files).
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We thank C. Bispo and A. Carlos (UCSF Flow Cytometry core facility) for excellent technical assistance. The work was supported by the UCSF Program for Breakthrough Biomedical Research and Sandler Foundation, March of Dimes grant no. 1-FY18-394 and 2018 AGA-Rome Foundation Functional GI and MotilityDisorders Pilot Research Award to F.F., the New York State Stem Cell Science (NYSTEM) contract C32599GG and a grant from the National Institutes of Neurological Disorders and Stroke (NINDS) 5R01NS099270 to L.S.
The Memorial Sloan-Kettering Cancer Center has filed a patent application (CA3009509A1) on hPSC-derived ENC lineages for use in Hirschsprung’s disease, with L.S. and F.F. as inventors. L.S. is a co-founder and consultant of BlueRock Therapeutics.
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Key reference(s) using this protocol
Fattahi, F. et al. Nature 531, 105–109 (2016): https://doi.org/10.1038/nature16951
Tchieu, J. et al. Cell Stem Cell 21, 399–410 (2017): https://doi.org/10.1016/j.stem.2017.08.015
Integrated supplementary information
KSR, knockout serum replacement differentiation medium; LDN, LDN-193189, SB, SB431542, CHIR, CHIR 99021; RA, Retinoic Acid; SB, SB431542.
Supplementary Figure 2 Representative phase contrast image of WA09 embryonic stem cells cultured in E8 medium.
Scale bar = 100 μm.
Supplementary Figure 3 Representative phase contrast images of differentiating cells at different time points of EN induction.
Scale bar = 200 μm.
a) Immunofluorescence staining of NOS1 and CHAT on day 75 of EN induction. b) Flow cytometry analysis of NOS1 and CHAT expression on day 75 on EN induction. AF647, Alexa Fluor™ 647; AF488, Alexa Fluor™ 488. Scale bar = 20 μm.
a) Phase contrast image of low density regions of culture plates on day 75 of differentiation. Arrows point to flat non-neuronal contaminating cells. b) Immunofluorescence staining of EN cultures with SMA and TUJ1 on day 75 of differentiation. Scale bar = 100 μm in a and 200 μm in b.
Supplementary Figure 6 Example of FACS gating strategy for purification of CD49D+ ENCs on day 12 of differentiation.
a) Unstained control sample. b) Sample stained with CD49D.
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Barber, K., Studer, L. & Fattahi, F. Derivation of enteric neuron lineages from human pluripotent stem cells. Nat Protoc 14, 1261–1279 (2019). https://doi.org/10.1038/s41596-019-0141-y
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