A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes

Arabinda Mandal; Chioma Moneme; Bhanu P Tewari; Allan M Goldstein; Harald Sontheimer; Lily Cheng; Sean R Moore; Daniel Levin

doi:10.1016/j.jneumeth.2024.110144

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes

J Neurosci Methods. 2024 Apr 25:407:110144. doi: 10.1016/j.jneumeth.2024.110144. Online ahead of print.

Authors

Arabinda Mandal¹, Chioma Moneme¹, Bhanu P Tewari², Allan M Goldstein³, Harald Sontheimer², Lily Cheng¹, Sean R Moore⁴, Daniel Levin⁵

Affiliations

¹ Department of Surgery, University of Virginia, Charlottesville, VA, USA.
² Department of Neuroscience, University of Virginia, Charlottesville, VA, USA.
³ Department of Pediatric Surgery, Massachusetts General Hospital, Boston, MA, USA.
⁴ Department of Pediatrics, Division of Pediatric Gastroenterology Hepatology, and Nutrition, University of Virginia, Charlottesville, VA, USA. Electronic address: srm5u@virginia.edu.
⁵ Department of Surgery, University of Virginia, Charlottesville, VA, USA. Electronic address: dlevin@ETCH.com.

PMID: 38670535
DOI: 10.1016/j.jneumeth.2024.110144

Abstract

Background: The enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics.

New method: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation.

Results: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells.

Comparison with existing methods: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials.

Conclusion: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

Keywords: Action potential; Enteric nervous system (ENS); Enteric neurons; Longitudinal muscle myenteric plexus (LMMP); Myenteric plexus; Neural progenitor cells; Neural stem cells; Neuron subtypes; Neurosphere.

Grants and funding

K08 DK133673/DK/NIDDK NIH HHS/United States