Efficient Generation of Trunk Neural Crest and Sympathetic Neurons from Human Pluripotent Stem Cells Via a Neuromesodermal Axial Progenitor Intermediate

Abstract

The neural crest (NC) is a multipotent embryonic cell population that generates various cell types in an axial position-dependent manner. Cranial NC cells give rise to mesoectodermal derivatives, melanocytes, neurons, and glia whereas the vagal NC generates the enteric nervous system and trunk NC cells produce sympathetic neurons and neuroendocrine cells. An attractive approach for studying human NC biology and modeling NC-associated developmental disorders (neurocristopathies) involves the in vitro production of NC cells from human pluripotent stem cells (hPSCs). However, most conventional differentiation protocols generate predominantly cranial NC cells but fail to induce trunk NC cells. Here we describe a detailed protocol for the efficient in vitro generation of trunk NC cells and their derivatives from hPSCs. This relies on the induction of an intermediate cell population that exhibits neural and mesodermal potential, resembling the embryonic neuromesodermal progenitors, which generate the postcranial body axis in vivo. © 2019 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cells (hPSCs); neural crest; neuromesodermal progenitors; sympathetic neurons.

Figure 1. Overview of stages of differentiation protocol.

The first stage of differentiation involves the generation of neuromesodermal axial progenitors which are induced from hPSCs within the first three days of the protocol. Axial progenitors are replated into neural crest inducing conditions and over the course of five days, acquire a trunk neural crest identity. Trunk neural crest cells give rise to sympathoadrenal precursors after 4 days of priming in BMP and Sonic Hedgehog and with further neural maturation from day 12 onwards in neurotrophin containing media.

Figure 2. In vitro generation of neuromesodermal axial progenitors from hPSCs

(A) hPSCs are replated into N2B27 media containing the WNT agonist CHIR99021 and recombinant bFGF supplemented with Y-27632-dihydrochloride for the first day. During days 2 and 3 of differnetiation, cells are exposed to CHIR99021 and bFGF alone. (B) Representative phase contrast images (Top: low magnification, Bottom: High magnification) of cells at day one after plating and at day three at the end of the Axial Progenitor differentiation stage. (C) Immunofluoresence images showing the heterogeneous co-expression of the axial progenitor markers BRACHYURY and SOX2. (D) qPCR data showing the induction of indicated axial progenitor marker genes. Note the downregulation of the pluripotency associated gene NANOG.

Figure 3. In vitro differentiation of axial progenitors toward trunk neural crest cells

(A) Day three axial progenitors are replated into neural crest-inducing conditions for five days (from day three until day eight). (B) Representative phase contrast images at 10x and 40x showing cell density and morphology after axial progenitor replating at day 4. Cells should proliferate and be confluent at day 7. (C) Immunostaining analysis showing SOX10 and HOXC9 co-expressing cells which comprise the bulk of the cultures at day 8 of differentiation (D) qPCR analysis showing a gain of neural crest identity over the course of days three to eight as indicated by expression of SOX10, TFAP2α, PAX3 and PAX7.

Figure 4. In vitro differentiation of trunk neural crest toward sympathetic neurons

(A) Day 8 trunk neural crest cells are plated into sympathoadrenal priming conditions containing Recombinant BMP4 and Sonic Hedgehog and the small molecule agonist of Sonic Hedgehog pathway Purmorphamine. At day 12, media is changed to contain neurotrophins to promote further differentiation toward a sympathetic neuronal identity. (B) Phase contrast images showing the acquisition of a neuronal morphology at day 9 after trunk neural crest cells have been replated. (C) Flow cytometry analysis of PHOX2b:GFP expression showing high yield of PHOX2b positive putative sympathoadrenal precursors at d12. (D) Immunofluorescence analysis of cells at day 19 after neuronal maturation showing the expression of sympathetic neuron markers GATA3, ISLET1/2, ASCL1 and the catecholamine synthesizing enzyme Tyrosine Hydroxylase.