Expansion of human mesothelial progenitor cells in a longterm three-dimensional organotypic culture of Processus vaginalis peritonei.

ABSTRACT

A 3D culture system was used to investigate the behaviour of mesothelial cells present in the wall of human processus vaginalis peritonei. Small tissue fragments placed on collagen sponges were cultured for 7, 14 and 21 days in medium supplemented with 10% FBS, and analysed for the expression and distribution of cytokeratins (CKAE1-AE3, CK19), p63, Ki-67, vimentin, CD34, and HBME-1. Before culture, flat mesothelial cells displayed immunoreactivity for cytokeratins, vimentin and HBME-1, while p63 and CD34 were negative. Mesenchymal cells within the stroma were vimentin-positive and endothelial cells of small vessels displayed positive staining for CD34. Cytokeratins, p63 and HBME-1 were negative in all stromal cells. In cultured fragments, flat mesothelial cells positive for vimentin, cytokeratins and HBME-1 proliferated, lining the fragment surface and migrating into the sponge. Capillaries showed morphological alterations; however, their immunoreactivity was comparable with the stroma prior to culture. Cells that had migrated into the sponge and displayed characteristics of mesothelial progenitors, predominantly spindleshaped and stellate, showed heterogeneous expression of markers especially in late phases of cultivation. These cells were constantly positive for vimentin, a small fraction was cytokeratin-positive and a few displayed HBME-1 immunoreactivity. CD34 was found in cells forming small cavities into the matrix, resembling newly formed blood vessels. Cells that had migrated into the sponge could be isolated and expanded in coculture with feeder NIH.3T3 fibroblasts. This system is suitable for studying growth and behaviour of mesothelial cells within their natural environment, providing a good method for isolation and expansion of their progenitor cells.