Understanding the Biology of Human Interstitial Cells of Cajal in Gastrointestinal Motility

Abstract

Millions of patients worldwide suffer from gastrointestinal (GI) motility disorders such as gastroparesis. These disorders typically include debilitating symptoms, such as chronic nausea and vomiting. As no cures are currently available, clinical care is limited to symptom management, while the underlying causes of impaired GI motility remain unaddressed. The efficient movement of contents through the GI tract is facilitated by peristalsis. These rhythmic slow waves of GI muscle contraction are mediated by several cell types, including smooth muscle cells, enteric neurons, telocytes, and specialised gut pacemaker cells called interstitial cells of Cajal (ICC). As ICC dysfunction or loss has been implicated in several GI motility disorders, ICC represent a potentially valuable therapeutic target. Due to their availability, murine ICC have been extensively studied at the molecular level using both normal and diseased GI tissue. In contrast, relatively little is known about the biology of human ICC or their involvement in GI disease pathogenesis. Here, we demonstrate human gastric tissue as a source of primary human cells with ICC phenotype. Further characterisation of these cells will provide new insights into human GI biology, with the potential for developing novel therapies to address the fundamental causes of GI dysmotility.

Keywords: ICC; bioinformatics; gastrointestinal motility; human; interstitial cells of Cajal; molecular characterisation; mouse; peristalsis; pluripotent stem cells..

Figure 1

Identification and capture of candidate interstitial cells of Cajal (ICC) from human gastric muscle tissue. (A) Outline of the experimental workflow for processing human sleeve gastrectomy samples for immunofluorescence or flow cytometry. (B–E) Immunofluorescence data obtained from cryosections of human gastric muscle stained with DAPI nuclear stain (blue) and antibodies that detect ICC marker proteins ANO1 (B; green) and KIT (C; red), with overlapping expression observed (D; ANO1+KIT+DAPI). A representative negative control (E; secondary antibody only control+DAPI) shows no background staining, thereby supporting the specificity of the staining patterns seen in B–D. Images were taken at 40× objective on the Zeiss Axio Imager M2 microscope. Scale bar = 50 μm. (F) A representative fluorescence-activated cell sorting (FACS) plot illustrating key human sleeve gastrectomy cell populations including: KIT⁺/CD45^-/CD11b^- ICC (red box); hematopoietic cells (HP; KIT^-/CD45⁺/CD11b⁺) (grey box); and mast cells (MC; KIT⁺/CD45⁺/CD11b⁺) (grey box). (G) Plot of ICC frequency and total sorted ICC numbers. Each dot represents an individual patient sample. (H) RT-PCR verification of cell marker genes in the FACS-captured ICC population: ANO1 (ICC), KIT (ICC, mast cells), CPA3 (mast cells), and CD68 (hematopoietic cells). Data were normalised against GAPDH mRNA levels, expressed relative to unsorted cells (dotted line), and represented as mean fold change ± standard error of the mean from three biological samples. Data were analysed using paired t-test; * p < 0.05, ** p < 0.01.

Figure 2

Workflow for characterisation of patient-derived human gastric interstitial cells of Cajal (ICC).