Derivation of enteric neuron lineages from human pluripotent stem cells

Abstract

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.

Figure 1. Major subtypes of the embryonic NC along the anterior to-posterior axis.

Migratory ENC progenitors are primarily derived from the vagal NC.

Figure 2. Induction of ENC cells from hPSCs.

a) Protocol (days 0–12) for ENC induction using option B. BMP4, Recombinant human bone morphogenetic protein-4; CHIR, CHIR 99021; RA, Retinoic Acid; SB, SB431542. b) Confluency of hPSCs on day 0 of differentiation. c) Phase contrast and SOX10::GFP reporter line GFP expression on day 2, day 6 and day 12. d) Representative image of FACS analysis of CD49D/SOX10::GFP positive ENC cells on day 12. e) Quantitative reverse transcriptase PCR (qRT–PCR) for vagal NC markers HOXB3, HOXB5, and ENC lineage marker PAX3 for ENC cells versus hPSCs. N=3 biological replicates. FC, fold change. Scale bars = 200 μm.

Figure 3. ENC spheroid culture.

a) Protocol (days 12–15) for ENC spheroid formation. NB+N2+B27; NB/N2/B27, Neurobasal medium with N2 and B27 supplement; FGF2, Recombinant Human FGF Basic; CHIR, CHIR 99021. b) Phase contrast and SOX10::GFP reporter line GFP expression of 3D spheroids on day 14. Scale bar = 200 μm.

Figure 4. Characterization of hPSC-derived ENC and enteric neurons.

a) Protocol for neuronal differentiation and maturation of ENC precursors, b) Flow cytometry analysis of CD49D positive ENC cells from hESC line UCSF4 and hiPSC line WTC11 on day 12. c) Flow cytometry analysis of CD49D positive ENC cells from hESC line UCSF4 and hiPSC line WTC11 after ENC spheroid enrichment on day 15. d) Immunofluorescence staining of TUJ1/TRKC on day 30 of EN induction. e) Flow cytometry analysis of TUJ1 and TRKC expression in EN cells on day 20, day 40 and day 55. f) Immunofluorescence images of CHAT, 5HT, NOS1, and GABA stained ENs on day 50. g) Flow cytometry analyses of CHAT, 5HT, NOS1, and GABA on ENS at day 75. AA, ascorbic acid; GDNF, Recombinant Human Glial Derived Neurotrophic Factor, F647, Alexa Fluor™ 647. Scale bars = 100 μm in c, f and 20 μm in e. o

Figure 5. Expression of glial lineage markers hPSC-derived EN population.

a) Immunofluorescence image of TUJ1/GFAP stained differentiated cultures on day 55. b) Flow cytometry analysis of SOX10 and GFAP expression on day 75 of differentiation. AF647, Alexa Fluor™ 647; AF488, Alexa Fluor™ 488.

Figure 6. Gene expression analysis of hPSC-derived enteric neurons.

a-f) Quantitative reverse transcriptase PCR (qRT–PCR) of ENS lineage markers PHOX2B, EDNRB, ASCL1, TUJ1, CHAT and GFAP for EN populations versus hPSCs. N=3 biological replicates. FC, fold change.

Figure 7. FACS purification of ENC lineages.

Time course flow cytometry analysis of CD49D expression in unsorted differentiated cultures (a) and populations sorted at day 11 for CD49D (b). FSC, forward scatter; SSC, side scatter.