A Model to Study the Phenotypic Changes of Interstitial Cells of Cajal in Gastrointestinal Diseases

Background & Aims

Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions.

Methods

Kit^+/copGFP mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP⁺ ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP⁺ ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit^+/copGFP mice were crossbred with diabetic Lep^+/ob mice to generate compound Kit^+/copGFP;Lep^ob/ob mutant mice. copGFP⁺ ICC from compound transgenic mice were analyzed by confocal microscopy.

Results

copGFP in Kit^+/copGFP mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP⁺ cells from jejunal muscles were Kit⁺ and free of contaminating cell-specific markers. Kit^+/copGFP;Lep^ob/ob mice displayed ICC networks that were dramatically disrupted during the development of diabetes.

Conclusions

Kit^+/copGFP mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Keywords: Kit, Gastrointestinal Tract, Diabetes, copGFP

Abbreviations used in this paper: 7-AAD, 7-amino-actinomycin D, Bio, biotin, ES, embryonic stem, Fc block, anti-mouse CD16/32, ICC, interstitial cells of Cajal, ICC-IM, intramuscular ICC, ICC-MY, myenteric ICC, ICC-SM, submucosal ICC, KI, knock-in, KIT, tyrosine kinase, PC7, PE-cyanine 7 tandem, PCR, polymerase chain reaction, PE, R-phycoerythrin, SA, streptavidin, TxR, Texas Red, eGFP, enhanced green fluorescent protein, ZsGree, Zoanthus sp. green fluorescent protein