Gastroenterology

Gastroenterology

Volume 134, Issue 5, May 2008, Pages 1424-1435
Gastroenterology

Basic–Alimentary Tract
Characterization of Fetal and Postnatal Enteric Neuronal Cell Lines With Improvement in Intestinal Neural Function

https://doi.org/10.1053/j.gastro.2008.02.018Get rights and content

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.

Section snippets

Reagents

Collagenase and dispase was purchased from Worthington Biochemical (Freehold, NJ), neurobasal-A medium and B-27 serum-free supplement was purchased from Invitrogen (Carlsbad, CA), recombinant Ms interferon-γ was purchased from Chemicon (Temecula, CA), the RNeasy mini kit was purchased from Qiagen (Stanford, CA), the iScript complementary DNA (cDNA) Synthesis kit and SYBR Green I were purchased from Bio-Rad (Hercules, CA), polymerase chain reaction (PCR) primers were purchased from IDT

Establishment of Cell Lines

Fetal cell lines were established from E-13 heterozygous immortomice. Postnatal cell lines were established from homozygous immortomice pups (1–2 days). The entire stomach and intestine were used to generate the cell lines. In the initial stages of the establishment of the lines we used 4-week-old homozygous or heterozygous immortomice and were not able to obtain cells that would divide and attain confluence. The first 10 mice that were used for the establishment of fetal cell lines did not

Discussion

By using the H-2Kb-tsA58 transgenic mice we have developed fetal and postnatal enteric neuronal cell lines with features similar to primary enteric neurons. The IM-FEN and IM-PEN have been passaged 40 times since their isolation and have shown no major changes in their cell growth characteristics, morphology, neuronal properties, or GDNF sensitivity. This homogeneity suggests that the lines will tend to remain phenotypically and biologically stable in vitro for many generations. One of the

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  • Cited by (0)

    Supported by National Institutes of Health grants KO8 DK067045 (S.S.), DK062092 (F.A.), DK075397 (F.A.), and DK06411 (S.V.S.), and the DDRDC (DK064399).

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