Single-cell RNA sequencing predicts motility networks in purified human gastric interstitial cells of Cajal

Background: Gastrointestinal (GI) motility disorders affect millions of people worldwide, yet they remain poorly treated in part due to insufficient knowledge of the molecular networks controlling GI motility. Interstitial cells of Cajal (ICC) are critical GI pacemaker cells, and abnormalities in ICC are implicated in GI motility disorders. Two cell surface proteins, KIT and ANO1, are used for identifying ICC. However, difficulties accessing human tissue and the low frequency of ICC in GI tissues have meant human ICC are insufficiently characterized. Here, a range of characterization assays including single-cell RNA sequencing (scRNA-seq) was performed using KIT+CD45−CD11B− primary human gastric ICC to better understand networks controlling human ICC biology. Methods: Excess sleeve gastrectomy tissues were dissected; ICC were analyzed by immunofluorescence, fluorescence-activated cell sorting (FACSorting), real-time PCR, mass spectrometry, and scRNA-seq. Key Results: Immunofluorescence identified ANO1+/KIT+ cells throughout the gastric muscle. Compared to the FACSorted negative cells, PCR showed the KIT+CD45−CD11B− ICC were enriched 28-fold in ANO1 expression (p < 0.01). scRNA-seq analysis of the KIT−CD45+CD11B+ and KIT+CD45−CD11B− ICC revealed separate clusters of immune cells and ICC (respectively); cells in the ICC cluster expressed critical GI motility genes (eg, CAV1 and PRKG1). The scRNA-seq data for these two cell clusters predicted protein interaction networks consistent with immune cell and ICC biology, respectively. Conclusions & Inferences: The single-cell transcriptome of purified KIT+CD45−CD11B− human gastric ICC presented here provides new molecular insights and hypotheses into evolving models of GI motility. This knowledge will provide an improved framework to investigate targeted therapies for GI motility disorders