Gunjan Tiwari

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

ROCK1/2 Inhibitor Induces Enteric Phenotype in CNS Derived Neural Stem Cells

Introduction Central nervous system-derived neural stem cells (CNS-NSC) have been previously shown to populate the enteric nervous system after transplantation in the gut. CNSNSC, are therefore considered promising candidates for cell based therapy for enteric neuropathies such as achalasia and Hirschsprungs disease. However the optimal conditions leading to the most efficient conversion of CNS-NSC to an enteric phenotype are still being investigated. Here we describe a simple and efficient method of inducing enteric phenotype in CNSNSC using the ROCK1/2 inhibitor Y27632. Methods CNS-NSC isolated from the subventricular zone of E13.5 mouse embryos were cultured under proliferating conditions in presence or absence of 25uM Y27632 and the expression of enteric markers was examined by qRT-PCR after 5 days. Results The CNS-NSC cultured in presence of Y27632 formed neurospheres that adhered and cells started migrating out of them and growing neural processes. On the other hand, CNS-NSC cultured in the absence of Y27632 continued to proliferate and grow as floating neurospheres. qRT-PCR analysis of cells cultured with Y27632 revealed 2.5-fold increase in the expression of p75, an enteric neural progenitor marker and 4.5 fold increase in the expression of Ret, a key regulator of enteric neuronal development over cells cultured without Y27632. The expression of Sox 10, a glial cell marker, was however found to be 3.3 fold lower in cells cultured with Y27632, suggesting preferential differentiation of CNS-NSC into neurons in presence of Y27632. Conclusions Inhibition of ROCK1/2 is a novel, highly efficient and less manipulative strategy for inducing enteric phenotype in NSC derived from CNS and it can potentially be expanded to induce enteric phenotype on stem cells of diverse origin.

Gene Expression Analysis of the Enteric Nervous System in Germ Free Mice

of βIII-tubulin (2-fold), ChAT (144-fold), NOS1 (66-fold), VIP (203-fold), and GFAP (748fold), compared to SMP, while those from the SMP layer formed more smooth muscle cells with an 11-fold increase in mRNA expression of ACTG2 (smooth muscle actin) compared to LM-MP. The proliferating neurospheres from LM-MP expressed substantially higher levels of neural crest markers p75 (157-fold) and Sox10 (142-fold), and neural progenitor marker, Nestin (3-fold). The same expression pattern was also present after sorting for Nes-Gfp + cells from the two layers, suggesting intrinsic differences in the stem cells rather than heterogeneity explains the observed phenotye. Upon differentiation, ENPs from aged mice were capable of differentiating into neurons, glial cells and smooth muscle cells, but formed less cholinergic neurons (3-fold less ChAT expression) than those from young mice. Conclusion: We find that spatial and temporal variables do affect ENP behavior.