Characterization of Fetal and Postnatal Enteric Neuronal Cell Lines With Improvement in Intestinal Neural Function

Background & Aims: The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2Kb-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 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 postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33°C, and interferon-γ for 24 or 48 hours, and then transferred to 39°C in the presence of glial cell line-derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, 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-hydroxytryptamine-receptor subtypes at 33°C and 39°C. Nestin, S-100β, and α-smooth muscle actin were expressed minimally at 39°C. Glial cell line-derived neurotrophic factor 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.