Clonal heterogeneity in murine liver myofibroblasts

GR primary cells cultures were isolated from hepatic granulomas induced in C3H mice livers by Schistosoma mansoni infection; the GRX continuous cell line was derived from GR cells after long-term culture and a progressive drift towards a rapidly proliferating cell population. These cells were analyzed and compared in terms of their clonal heterogeneity. Clones were classified on the basis of cell substrate, cell-cell adhesion (growth morphology of the clone) and fat droplet accumultation. GR cells were composed of two slow-growing clone types, while GRX cells grave rise to clones with several phenotypes, including the two found in the GR cells. The overall proportion of different clones in the GRX cell population was stable in long-term cultures, as well as after recloning of the highly proliferating, but not the slowly proliferating, clones. We propose that the slow-growing clones are maintained in the overall population by continuous contribution of new slow-growing cells from the rapidly growing ones. The slow-growing clones may represent the basal population of liver connective tissue cells that can be mobilized into injured tissues and that are involved in tissue repair. The highth proliferating clones with a broad capacity of phenotype expression that arise after long-term growth simulation of the local cell population may represent the hypertrophic connective tissue cells, such as those observed in progressive fibrotic reactions associated with chronic liver tissue inflammation

In vitro collagen synthesis by liver connective tissue cells isolated from schistosomal granulomas

Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.