Characterization and transplantation of enteric neural crest cells from human induced pluripotent stem cells

Abstract

The enteric nervous system (ENS) is recognized as a second brain because of its complexity and its largely autonomic control of bowel function. Recent progress in studying the interactions between the ENS and the central nervous system (CNS) has implicated alterations of the gut/brain axis as a possible mechanism in the pathophysiology of autism spectrum disorders (ASDs), Parkinson’s disease (PD) and other human CNS disorders, whereas the underlying mechanisms are largely unknown because of the lack of good model systems. Human induced pluripotent stem cells (hiPSCs) have the ability to proliferate indefinitely and differentiate into cells of all three germ layers, thus making iPSCs an ideal source of cells for disease modelling and cell therapy. Here, hiPSCs were induced to differentiate into neural crest stem cells (NCSCs) efficiently. When co-cultured with smooth muscle layers of ganglionic gut tissue, the NCSCs differentiated into different subtypes of mature enteric-like neurons expressing nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), choline acetyltransferase (ChAT) or calretinin with typical electrophysiological characteristics of functional neurons. Furthermore, when they were transplanted into aneural or aganglionic chick, mouse or human gut tissues in ovo, in vitro or in vivo, hiPSC-derived NCSCs showed extensive migration and neural differentiation capacity, generating neurons and glial cells that expressed phenotypic markers characteristic of the enteric nervous system. Our results indicate that enteric NCSCs derived from hiPSCs supply a powerful tool for studying the pathogenesis of gastrointestinal disorders and brain/gut dysfunction and represent a potentially ideal cell source for enteric neural transplantation treatments.

Cell therapy for GI motility disorders: comparison of cell sources and proposed steps for treating Hirschsprung disease

“…In another recent study, Li et al (46) demonstrated the efficient generation of enteric neural progenitors from human PSCs using an alternative induction protocol to Fattahi et al (22) involving embryoid body formation and Rho kinase (ROCK) inhibition, similar to that previously described. Engraftment and differentiation of PSC-derived enteric neural progenitors were shown in explants of colonic smooth muscle from Hirschsprung disease patients (46).…”

“…In another recent study, Li et al (46) demonstrated the efficient generation of enteric neural progenitors from human PSCs using an alternative induction protocol to Fattahi et al (22) involving embryoid body formation and Rho kinase (ROCK) inhibition, similar to that previously described. Engraftment and differentiation of PSC-derived enteric neural progenitors were shown in explants of colonic smooth muscle from Hirschsprung disease patients (46). Transplantation into mouse distal colon was also performed, resulting in migration of 4 -8 mm, which is more consistent with a previously published study using endogenous enteric neural progenitor (35), although the number of progenitors transplanted by Li et al (2016) was not reported and is likely to play a major role in engraftment outcomes.…”

“…Until recently though, protocols to obtain large numbers of enteric neural progenitors from PSCs had not been developed. However, several recent studies have provided evidence of efficient generation of enteric neural progenitors from human PSCs, which were utilized for drug screening, in vitro disease modeling, and successful transplantation into Hirschsprung disease model mice (22,46,69).…”

“…Cell therapy requires an accessible source of ENS progenitor cells to transplant into the bowel. Three recent studies have demonstrated the generation of enteric neural progenitors from human pluripotent stem cells (PSCs), utilizing them for transplantation in small animal models, or for in vitro modeling of Hirschsprung disease (22,46,69). Fattahi et al (22) and Li et al (46) showed that enteric neural precursors could be generated from human PSCs, and following transplantation into the colon of adult mice, the precursors migrated and generated enteric neurons.…”

Enteric neural stem cell therapies for enteric neuropathies

“…This study also showed the ability of these human iPSC‐derived NCSC to colonize aneural mouse and chick gut. Furthermore, engraftment of iPSC‐derived NCSC within BAC‐treated ganglionic and aganglionic gut tissue from human HSCR patients led to the development of enteric‐like neurons and reductions in contractile frequency in ex vivo cultures . However, it is unclear how many iPSC‐derived NCSC were transplanted in these conditions making any comparison difficult.…”