Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.
OBJECTIVE
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-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) 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 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees 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 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees 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.
Anitha
Mallappa
M
Division of Digestive Diseases, Emory University, Atlanta, Georgia, USA.
Joseph
Irene
I
Ding
Xiaokun
X
Torre
Enrique R
ER
Sawchuk
Michael A
MA
Mwangi
Simon
S
Hochman
Shawn
S
Sitaraman
Shanthi V
SV
Anania
Frank
F
Srinivasan
Shanthi
S
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003106:Colon / Q000196:embryology / Q000294:innervation* / Q000601:surgery; D051959:ELAV Proteins / Q000096:biosynthesis / Q000235:genetics; D000067779:ELAV-Like Protein 4; D017615:Enteric Nervous System / Q000196:embryology* / Q000378:metabolism; D005260:Female; D005769:Gastrointestinal Motility / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000235:genetics; D007537:Isometric Contraction / Q000502:physiology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009130:Muscle, Smooth / Q000294:innervation / Q000502:physiology; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D064231:Nestin; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D064531:Peripherins; D011247:Pregnancy; D051096:Proto-Oncogene Proteins c-ret / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000096:biosynthesis / Q000235:genetics; D012701:Serotonin / Q000096:biosynthesis / Q000235:genetics; D016708:Synaptophysin / Q000096:biosynthesis / Q000235:genetics; D043222:Ubiquitin Thiolesterase / Q000096:biosynthesis / Q000235:genetics; D029867:Xenopus Proteins; D016875:tau Proteins / Q000096:biosynthesis / Q000235:genetics
D000199:Actins; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C498011:Elavl4 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; C578995:PRPH protein, Xenopus; D064531:Peripherins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D016708:Synaptophysin; D029867:Xenopus Proteins; D016875:tau Proteins; D012701:Serotonin; D012313:RNA; D051096:Proto-Oncogene Proteins c-ret; C494999:Ret protein, mouse; D043222:Ubiquitin Thiolesterase; C496976:Uchl1 protein, mouse