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

Abstract

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+/obmice to generate mice compound Kit+/copGFP;Lepob/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; Lepob/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.

Transcriptome of interstitial cells of Cajal reveals unique and selective gene signatures

“…CopGFP-labeled cells within jejunal smooth muscle layers were identified by confocal microscopy (Fig 1A), and these cells were confirmed to be Kit + ICC by immunohistochemical labeling with KIT antibodies (Fig 1B), as previously reported [13]. ICC within the plane of the myenteric plexus (ICC-MY) and the deep muscular plexus (ICC-DMP) were labeled by copGFP (labeling of cytoplasm) and KIT antibodies (labeling of plasma membrane) (Fig 1C).…”

“…RT-PCR and qPCR analyses of cDNA were performed as previously described [13, 18]. All primers used for RT-PCR and qPCR are shown in S11 Table.…”

Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1

“…Kit +/copGFP (B6.129S7- Kit tm1Rosay /J) mice expressing copepod green fluorescent protein (copGFP) from the endogenous Kit locus, 21 Pdgfra +/eGFP (B6.129S4- Pdgfra tm11(EGFP)Sor /J) mice expressing histone H2B-enhanced GFP (eGFP) fusion protein from the endogenous Pdgfra locus 22 and strain-matched wild-type mice were from The Jackson Laboratory (Bar Harbor, ME). C57BL/6J, WBB6F1/J, WCB6F1/J and NOD/ShiLtJ mice 8 were also from The Jackson Laboratory.…”

c-kit+ cells minimally contribute cardiomyocytes to the heart

“…It was previously shown that knock-down of the Kit gene results in defective progenitor cell activity in many tissues 19–22 . Indeed, hearts from Kit w/wv mice showed a dramatic reduction in resident mononuclear c-kit + cells and progenitor activity 23 .…”