Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos

Abstract

SUMMARYNeural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors. Sox10-GFP-positive cells are produced transiently from mouse embryonic stem (ES) cells by treatment with retinoic acid in combination with Fgf8b and the cytokine leukaemia inhibitory factor (Lif). We found that expression of Sox10 can be maintained using noggin, Wnt3a, Lif and endothelin 3 (NWLE). ES cell-derived Sox10-GFP-positive cells cultured in NWLE exhibit molecular markers of neural crest progenitors. They differentiate into peripheral neurons in vitro and are able to colonise the enteric network in organotypic gut cultures. Neural crest cells purified from embryos using the Sox10 reporter also survive in NWLE, but progressively succumb to differentiation. We therefore applied selection to eliminate differentiating cells. Sox10-selected cells could be clonally expanded, cryopreserved, and multiplied for over 50 days in adherent culture. They remained neurogenic in vitro and in foetal gut grafts. Generation of neural crest from mouse ES cells opens a new route to the identification and validation of determination factors. Furthermore, the ability to propagate undifferentiated progenitors creates an opportunity for experimental dissection of the stimuli and molecular circuitry that govern neural crest lineage progression. Finally, the demonstration of robust enteric neurogenesis provides a system for investigating and modelling cell therapeutic approaches to neurocristopathies such as Hirschsprung's disease.

Neural crest stem cells: discovery, properties and potential for therapy

“…Recently, a combination of factors that promote self-renewal of these progenitors resulting in their significant expansion as undifferentiated NCCs was defined [76]. A Sox10-GFP transgene was used to isolate mESCs positive for GFP and these cells expressed NCC markers when cultured.…”

Neural crest cells retain their capability for multipotential differentiation even after lineage‐restricted stages

“…In this case, the Sox10-Venus-marked cells in Sox10-IRES-Venus mice should accurately reflect the behavior of the wildtype NC cells. Recently, chimeric mice generated from ES cells in which exon I of the Sox10 had been replaced by the GFP sequence were also reported to mark NC cells in vivo (Kawaguchi et al, 2010).…”

Transplanted progenitors generate functional enteric neurons in the postnatal colon

“…Although there are a number of possible sources of enteric neurons (4,5,7,8,10,11,13,16,(34)(35)(36)(37)(38)(39), enteric neural crest-derived ENS progenitors were chosen, as they are likely to be the most clinically relevant source of cells, are readily accessible (13), and can give rise to enteric neurons in the embryonic gut or when cocultured with colonic muscle from infants (13,14,18). We showed that after transplantation into the colon of postnatal mice, ENS progenitors proliferated; migrated extensively and differentiated into neurons with the neurochemical, morphological, and electrophysiological characteristics of enteric neurons; and received synaptic inputs.…”