Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers, and mediators of neurotransmission in the gastrointestinal tract

Abstract

Background Interstitial cells of Cajal (ICCs) are mesenchymal cells that play critical roles in gastrointestinal motility as electrical pacemakers and mediators of neuromuscular neurotransmission. Although depletions of ICCs have been implicated in several gastrointestinal motor disorders, quantification of these cells has been difficult due to their varied morphology, regionally changing network density, and overall scarcity. Our goal was to evaluate flow cytometry (FCM) for the enumeration of ICCs. Methods We identified murine ICCs in live gastrointestinal muscles or primary cell cultures grown in the presence or absence of stem cell factor (SCF)–expressing STO fibroblasts with fluorescent Kit (CD117) antibodies. Because this technique also labels resident macrophages nonspecifically, we identified the latter with additional fluorescent antibodies. Dispersed cells were analyzed by FCM. Results ICCs represented 1.63 ± 0.17% of the total cell count in the distal stomach (n = 18 mice) and 5.85 ± 0.84% in the proximal colon and 6.28 ± 0.61% in the distal colon (n = 3 mice). In fundic muscles of W/W^V mice (n = 5) that virtually lack ICCs, very few Kit⁺ cells were detected. FCM identified approximately 2.6‐ to 7.3‐fold more Kit⁺ ICCs in small intestinal cell cultures grown on STO fibroblasts expressing membrane‐bound SCF (n = 6) than in cultures stimulated with soluble SCF (n = 6). Conclusions FCM is a sensitive and specific method for the unbiased quantification of ICCs.