Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function

• Authors: Mallappa Anitha, Emory University; Irene Joseph, Emory University; Xiaokun Ding, Emory University; Enrique Torre, Emory University; Michael A Sawchuk, Emory University; Simon Mwangi, Emory University; Shawn Hochman, Emory University; Shanthi V. Sitaraman, Emory University; Frank A Anania, Emory University; Shanthi Srinivasan, Emory University
• Journal Title: Gastroenterology
• Volume: Volume 134, Number 5
• Publisher: Elsevier: 12 months | 2008-05, Pages 1424-1435
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1053/j.gastro.2008.02.018
• Subjects: Biology, Neuroscience; Health Sciences, Medicine and Surgery
• Keywords: Immorto; Enteric neurons; GDNF; proliferation; differentiation; Piebald; None

Abstract

Background and Aims The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal (IM-FEN) and postnatal (IM-PEN) enteric neuronal cell lines using the H-2Kb-tsA58 transgenic mice that have a temperature sensitive mutation of the SV-40 large tumor antigen gene under the control of an interferon γ-inducible H-2Kb promoter element. Methods Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day post natal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33°C and IFN-γ for 24 or 48 h and then transferred to 39°C in the presence of GDNF for 7 days for further differentiation. Neuronal markers were assessed by RT-PCR, western blot and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS−/− mice. Results Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, Tau, synaptic marker Synaptophysin, characteristic receptors of enteric neurons, Ret and 5-HT receptor subtypes at 33°C and 39°C. Nestin, S-100β and α-SMA were minimally expressed at 39°C. GDNF resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS−/− mice colon improved colonic motility. Conclusions We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung’s disease.