Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

Abstract

ObjectivesEnteric neural stem cells provide hope of curative treatment for enteric neuropathies. Current protocols for their harvesting from humans focus on the generation of ‘neurospheres’ from cultures of dissociated gut tissue. The study aims to better understand the derivation, generation and composition of enteric neurospheres.DesignGut tissue was obtained from Wnt1-Cre;Rosa26Yfp/Yfp transgenic mice (constitutively labeled neural crest cells) and paediatric patients. Gut cells were cultured either unsorted (mixed neural crest/non-neural crest), or following FACS selection into neural crest (murine-YFP+ve/human-p75+ve) or non-neural crest (YFP-ve/p75-ve) populations. Cultures and resultant neurospheres were characterized using immunolabelling in vitro and following transplantation in vivo.ResultsCultures of (i) unsorted, (ii) neural crest, and (iii) non-neural crest cell populations generated neurospheres similar in numbers, size and morphology. Unsorted neurospheres were highly heterogeneous for neural crest content. Neural crest-derived (YFP+ve/p75+ve) neurospheres contained only neural derivatives (neurons and glia) and were devoid of non-neural cells (i.e. negative for SMA, c-Kit), with the converse true for non-neural crest-derived (YFP-ve/p75-ve) ‘neurospheres’. Under differentiation conditions only YFP+ve cells gave rise to neural derivatives. Both YFP+ve and YFP-ve cells displayed proliferation and spread upon transplantation in vivo, but YFP-ve cells did not locate or integrate within the host ENS.ConclusionsSpherical accumulations of cells, so-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for ENS cell replacement therapy then improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, non-ENS cells.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…Depending on the individual culture protocols and markers used, NLBs from early postnatal mouse/rat gut are immunoreactive for typical neural differentiation markers after an in vitro culture period up to 2 weeks (Belkind-Gerson et al, 2013; Binder et al, 2015; Bondurand et al, 2003; Dettmann et al, 2014; Kruger et al, 2002; Silva et al, 2008). A small fraction of NLB cells (<15%) remain negative for all differentiation markers used, raising the possibility that at least a subfraction of the isolated cells that generate neurospheres are undifferentiated progenitors or NSCs (Bondurand et al, 2003).…”

“…Some investigators have attempted to enrich putative fetal and postnatal ENS progenitors using either surface markers such as RET (Binder et al, 2015; Lo and Anderson, 1995; Natarajan et al, 1999), the neurotrophin receptor p75(NTR), α4 integrin (Bixby et al, 2002; Kruger et al, 2002; Mosher et al, 2007; Tsai et al, 2011) or Sox2/Sox10/Nestin promoter-driven reporter genes (Bondurand et al, 2006; Heanue and Pachnis, 2011). Using these approaches only some of the selected cells proliferated and not all proliferating colonies were equally multipotent.…”

“…Using these approaches only some of the selected cells proliferated and not all proliferating colonies were equally multipotent. With respect to markers identifying differentiation, unpurified NLB cultures showed an increasing number of mature neurons and glia during in vitro culture and after about 2 weeks only 2% of cells remained negative for the pan-neural markers beta-tubulin III or the glial protein S100beta suggesting that the applied culture conditions support the in vitro differentiation of neurogenic and gliogenic progenitors (Binder et al, 2015). Further, qualitative immunostainings for neuronal subtypes could be demonstrated, which include markers for nitric oxide synthase (NOS1), choline acetyltransferase (CHAT), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), calcitonin gene-related peptide (CALCA or CGRP), tryptophan hydroxylase (TPH1) and tyrosine hydroxylase (TH); however especially TPH1+ (serotonergic) and TH+ (sympathoadrenal) neurons tend to decline with increasing age of donor tissue (Table 1; (Almond et al, 2007; Binder et al, 2015; Bondurand et al, 2003; Kruger et al, 2002).…”

Delivery of enteric neural progenitors with 5-HT4 agonist-loaded nanoparticles and thermosensitive hydrogel enhances cell proliferation and differentiation following transplantation in vivo

“…Most previous studies have demonstrated successful expansion of ENSCs as enteric “neurospheres.” In theory, dissociated intestinal cells are cultured in the appropriate conditions to allow neural progenitors to proliferate and other cell types to be eliminated spontaneously [13, 45, 46]. However, others have shown that enteric neurospheres consist of a heterogeneous population of cells, including neurons, glial cells, and non-neural crest-derived cells, such as smooth muscle and mesenchymal cells [8, 43]. We analyzed cell composition in our dissociated neurospheres and observed p75-immunoreactivity in up to 25% of cells, Tuj1+ neurons in 20%, and S-100+ glia in 12%.…”

Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic

“…Neurospherederived SMA + cells have been described by other groups using similar culture methods 20,37 and may represent either greater differentiation potential of the neurosphere progenitor cells or incomplete purification of the culture. Although the organization of these mesenchymal components is comparatively disorganized, this study has taken the initial steps to demonstrate that OU contain essential elements for mesenchymal regeneration, including neural elements.…”