Fibroblasts and their Precursors Can be Generated from Mesocolic Mesothelium; Implications for Intraperitoneal Fibrotic Diseases

Introduction: We recently identified a mesothelial layer overlying the entire mesocolon. Additionally, we identified a submesothelial connective tissue layer as well as sporadic fibroblast reservoirs beneath this monolayer. We speculated that these fibroblasts could be generated through ex vivo culture of the mesocolic mesothelium (MM). Aim: To develop a novel mechanism for ex vivo harvest and culture of MM to determine if this process could lead to fibrocyte and fibroblast generation. Method: Following ethical approval and informed consent, a technique was developed enabling the stripping and harvest of MM in patients undergoing colorectal resections. From these, a single cell suspension was prepared and cultured ex vivo. The cellular composition of the suspension was subsequently characterised via immunofluorescence and confocal microscopy with markers targeting collagen-1 (C), vimentin (V), Golgi apparatus (GM-130), phalloidin (P) and CD45. Following a period in culture, this was repeated. Results: Initial staining demonstrated mesothelial cells (V+ CD45-). Spindle-shaped cells morphologically resembling fibroblasts appeared in 9 (of 21) samples 10.5 (±13.3) days post plating. These were derived from a mixture of oncologic (n = 7), Crohns (n = 1) and diverticular (n = 1) resections. Dual staining with C and V confirmed these were mainly fibroblasts whilst dual staining with CD45 and C identified a fibrocyte subpopulation. Conclusion: MM can be harvested from benign and oncologic colorectal resections. This is predominantly mesothelial in composition at first. Following a period in culture, it is replaced by fibrocytes and fibroblasts. As MM thus represents a source of mesenchymal cells, these observations have implications for intraperitoneal fibrotic conditions