ABSTRACT
The peritoneum is a thin membrane that covers most of the abdominal organs, composed of a monolayer of mesothelial cells and subjacent submesothelial loose connective tissue. Cells from the peritoneal wall are correlated with peritoneal fibrosis and epithelial-to-mesenchymal transition. However, the distinct involvement of mesothelial or submesothelial cells in such phenomena is still not clear. Here, we propose a new strategy to obtain stromal cells from anterior peritoneal wall explant cultures. These cells migrated from peritoneal tissues and proliferated in vitro for 4 weeks as adherent fibroblast-like cells. Optical and electronic microscopy analyses of the fragments revealed a significant submesothelial disorganization. The obtained cells were characterized as cytokeratin- vimentin+ laminin+ α-smooth muscle actin+, suggesting a connective tissue origin. Moreover, at the third passage, these stromal cells were CD90+CD73+CD29+Flk-1+CD45-, a phenotype normally attributed to cells of mesenchymal origin. These cells were able to support hematopoiesis, expressing genes involved in myelopoiesis (SCF, G-CSF, GM-CSF, IL-7 and CXCL-12), and differentiated into osteogenic and adipogenic cell lineages. The methodology demonstrated in this work can be considered an excellent experimental model to understand the physiology of the peritoneal wall in healthy and pathological processes. Moreover, this work shows for the first time that submesothelial stromal cells have properties similar to those of mesenchymal cells from other origins.
Subject(s)
Adipogenesis , Cell Lineage , Epithelium/metabolism , Hematopoiesis , Osteogenesis , Peritoneum/cytology , Animals , Cell Movement , Cell Separation , Coculture Techniques , Flow Cytometry , Kinetics , Male , Mice, Inbred BALB C , Myelopoiesis , Peritoneum/ultrastructure , Phenotype , Stromal Cells/cytology , Stromal Cells/metabolismABSTRACT
In order to evaluate the effect of chaotropic agents on proteoglycan and non-collagenous proteins, chicken xiphoid cartilage was treated with guanidine-HCl and MgCl2 in different concentrations (1M to 5M), and different periods of time (12, 24, 48 and 72 hr). The maximum yield of uronic acid was obtained with 3M MgCl2 (73.3%). Concentrations of 4M and 5M of MgCl2 showed that much less uronic acid was removed, 55.3% and 38.1% respectively. Extraction with 3M MgCl2 and 3M guanidine-HCl resulted better efficiency when performed for 48 hr. Analysis by SDS-PAGE of the extracts obtained with guanidine-HCl and MgCl2 in different concentrations pointed out that most components are equally removed with the two solvents, showing that the extraction with MgCl2 is an alternative assay to remove non-collagenous proteins from extracellular matrix.
