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Updated: Feb 26, 2026
Isolation of Mouse Peritoneal Cavity Cells
Published on: January 28, 2010
Pure isolation, culture, and postinjury lineage tracing of mouse visceral pleural mesothelial cells
Akikazu Endo1, Yasunori Enomoto1, Ryo Horiguchi2
1Department of Regenerative and Infectious Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Abstract:
Pleural mesothelial cells (PMCs) are a major component of the pleura. The involvement of visceral PMCs has been suggested in several pleural or subpleural lung diseases, thus highlighting the importance of animal experiments using these cells. However, in wild-type rodent lungs, no direct and pure isolation method for visceral PMCs has been established so far. Using reanalyzed single-cell RNA sequencing data, we identified that mesothelin (Msln), a specifically expressed gene in visceral PMCs, can be useful for live cell sorting. After collecting cells by scraping the visceral pleura, MSLN+EpCAM-CD45-CD31-PDGFRα-CD146- cells with large size (≥1.4 times the median value of forward scatter in total cells) mostly exhibited WT1 protein (WT1 positivity in immunocytochemistry: 93.3% ± 0.8%). The sorted PMCs were culturable, and they responded generally predictably to several growth factors and cytokines, including genetic changes suggestive of a TGF-β-induced mesothelial-to-mesenchymal transition. In vivo experiments performed using PMC-specific reporter mice (Wt1-creERT2; tdTomato) demonstrated that intrapleural administration of bleomycin with carbon induced proliferation of PMCs in the visceral pleura. Importantly, some PMC-derived cells differentiated into α-smooth muscle actin-positive myofibroblasts with decreased MSLN expression, indicating that our method using MSLN is suitable only for uninjured lungs. In summary, we propose a novel and qualified method for visceral PMC isolation, which will aid in the elucidation of the mechanisms underlying pleura-related human lung diseases.
Insights
Researchers developed a new method to isolate visceral pleural mesothelial cells (PMCs) using mesothelin (MSLN) for studying lung diseases. This technique aids in understanding pleura-related conditions in animal models.
Area of Science:
- Cell Biology
- Pulmonary Medicine
- Immunology
Background:
- Pleural mesothelial cells (PMCs) are crucial for pleural health and implicated in lung diseases.
- Existing methods for isolating visceral PMCs in rodents are insufficient for research.
- Understanding PMC behavior is vital for studying pleural and subpleural lung pathologies.
Purpose of the Study:
- To establish a novel and efficient method for isolating pure visceral PMCs from wild-type rodent lungs.
- To characterize the isolated PMCs and assess their behavior in vitro and in vivo.
- To provide a tool for investigating the role of PMCs in pleura-related lung diseases.
Main Methods:
- Utilized single-cell RNA sequencing data to identify mesothelin (MSLN) as a specific marker for visceral PMCs.
- Developed a cell sorting strategy combining MSLN expression with other markers (EpCAM, CD45, CD31, PDGFRα, CD146) and cell size.
- Cultured sorted PMCs and induced TGFβ-mediated mesothelial-to-mesenchymal transition.
- Employed PMC-specific reporter mice to observe PMC proliferation and differentiation in vivo after bleomycin/carbon challenge.
Main Results:
- Successfully isolated and sorted visceral PMCs with high purity (93.3% WT1-positive).
- Sorted PMCs were viable, culturable, and responded to growth factors and cytokines.
- In vivo studies showed PMC proliferation in response to injury, with some differentiating into myofibroblasts.
- MSLN expression decreased upon differentiation, indicating the method's limitation to uninjured lungs.
Conclusions:
- A novel, qualified method for isolating visceral PMCs using MSLN has been developed.
- This method facilitates the study of PMC behavior in uninjured lungs.
- The findings provide a valuable tool for elucidating mechanisms in pleura-related human lung diseases.
