Isolation and gene expression profiling of intestinal epithelial cells: crypt isolation by calcium chelation from in vivo samples

Abstract

Aine Balfe,1,2 Grainne Lennon,1,2 Aonghus Lavelle,1,2 Neil G Docherty,1 J Calvin Coffey,3 Kieran Sheahan,4 Desmond C Winter,2 P Ronan O’Connell1,2 1School of Medicine and Medical Science, University College Dublin, Belfield, Dublin, 2Centre for Colorectal Disease, St Vincent’s University Hospital Dublin, Dublin, 3Graduate Entry Medical School, University Hospital Limerick, 4i Centre for Interventions in Infection, Inflammation and Immunity, University of Limerick, Limerick, 4Histopathology Department, St. Vincent’s University Hospital Dublin, Dublin, Ireland Aim: The epithelial layer within the colon represents a physical barrier between the luminal contents and its underlying mucosa. It plays a pivotal role in mucosal homeostasis, and both tolerance and anti-pathogenic immune responses. Identifying signals of inflammation initiation and responses to stimuli from within the epithelial layer is critical to understanding the molecular pathways underlying disease pathology. This study validated a method to isolate and analyze epithelial populations, enabling investigations of epithelial function and response in a variety of disease setting. Materials and methods: Epithelial cells were isolated from whole mucosal biopsies harvested from healthy controls and patients with active ulcerative colitis by calcium chelation. The purity of isolated cells was assessed by flow cytometry. The expression profiles of a panel of epithelial functional genes were investigated by reverse transcription-polymerase chain reaction (PCR) in isolated epithelial cells and corresponding mucosal biopsies. The expression profiles of isolated cells and corresponding mucosal biopsies were evaluated and compared between healthy and inflamed colonic tissue. Results: Flow cytometry identified 97% of cells isolated as intestinal epithelial cells (IECs). Comparisons of gene expression profiles between the mucosal biopsies and isolated IECs demonstrated clear differences in the gene expression signatures. Sixty percent of the examined genes showed contrasting trends of expression between sample types. Conclusion: The calcium chelation isolation method provided a reliable method for the isolation of a pure population of cells with preservation of epithelial cell-specific gene expression. This demonstrates the importance of sample choice when investigating functions directly affecting the colonic epithelial layer. Keywords: epithelial cells, ulcerative colitis, gene expression, mucosal biopsies, molecular pathways, colonic inflammation

Keywords

• Epithelial cells
• ulcerative colitis
• gene expression
• mucosal biopsies