Abstract
Enteric nervous system (ENS) progenitor cells isolated from mouse and human bowel can be cultured in vitro as neurospheres which are aggregates of the proliferating progenitor cells, together with neurons and glial cells derived from them. To investigate the factors regulating progenitor cell proliferation and differentiation, we first characterised cell proliferation in mouse ENS neurospheres by pulse chase experiments using thymidine analogs. We demonstrate rapid and continuous cell proliferation near the neurosphere periphery, after which postmitotic cells move away from the periphery to become distributed throughout the neurosphere. While many proliferating cells expressed glial markers, expression of the neuronal markers β-tubulin III (Tuj1) and nitric oxide synthase was detected in increasing numbers of post-mitotic cells after a delay of several days. Treatment of both mouse and human neurospheres with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced expression of the transcription factors Hes1 and Hes5, demonstrating inhibition of Notch signaling. DAPT treatment also inhibited progenitor cell proliferation and increased the numbers of differentiating neurons expressing Tuj1 and nitric oxide synthase. To confirm that the cellular effects of DAPT treatment were due to inhibition of Notch signaling, siRNA knockdown of RBPjκ, a key component of the canonical Notch signaling pathway, was demonstrated both to reduce proliferation and to increase neuronal differentiation in neurosphere cells. These observations indicate that Notch signaling promotes progenitor cell proliferation and inhibits neuronal differentiation in ENS neurospheres.
Highlights
During vertebrate embryonic development, enteric nervous system (ENS) progenitor cells arising primarily from the vagal region of the neural crest migrate rostrocaudally along the gut, proliferating and differentiating to form the ganglia of the ENS [1,2,3]
Neurosphere culture offers a potential method for the amplification of ENS progenitor cells for future transplantation therapies in Hirschsprung’s disease (HSCR) [6,8,13], there have been no studies into why the cells proliferate rapidly in neurospheres, nor why some of them cease proliferation and differentiate into postmitotic neural cells
Our investigations here demonstrate that rapid cell proliferation occurs at or near the periphery of ENS neurospheres, after which postmitotic cells move throughout the neurosphere
Summary
Enteric nervous system (ENS) progenitor cells arising primarily from the vagal region of the neural crest migrate rostrocaudally along the gut, proliferating and differentiating to form the ganglia of the ENS [1,2,3]. Failure of this migration in humans results in Hirschsprung’s disease (HSCR), characterised by intestinal aganglionosis, which typically extends to a variable extent rostrally to include the internal anal sphincter, rectum and distal colon [4]. We demonstrate using chemical and siRNA inhibition that Notch signaling is necessary both for the maintenance of cell proliferation and suppression of neuronal differentiation in ENS neurospheres
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