Nontransformed colony-derived stromal cell lines from normal human marrows. II. Phenotypic characterization and differentiation pathway

In a previous report, we described a method to generate cell lines derived from stromal colonies (colony-derived cell lines [CDCL]). In a first step, colonies were obtained from plating cells from adherent layers of human long-term bone marrow cultures (LTBMC) in methyl-cellulose in the presence of interleukin-1 beta (IL-1 beta) (20 U/mL) and tumor necrosis factor-alpha (TNF-alpha) (200 U/mL). In a second step, cell lines were derived from individual colonies cultured in liquid medium with 20 ng/mL basic fibroblast growth factor (bFGF). In this report, we describe the phenotype of cells from more than 100 cell lines. CDCL did not contain cells of hematopoietic origin, which indicates that the culture system did not allow the growth of hematopoietic precursors. CDCL did not contain endothelial-like cells, similarly to primary adherent layers. CDCL comprised stromal cells, as defined by membrane antigens recognized by monoclonal antibodies 6-19, Stro-1 and 1B10. These cells belonged to the family of connective tissue-forming cells since they synthesized interstitial collagens I, III, and V; nonplasmatic EDa+ and EDb- fibronectin; tenascin; and chondroitin-sulfate. Study of the time course of CDCL showed that the lines differed from one another according to their proliferative capacity: 32% of CDCL grew quickly, yielding about 10,000 cells in 10 days; 36% of CDCL grew slowly, yielding 1000 cells in 10 days; and the remaining 32% had intermediate proliferative capacity. For CDCL with a high proliferative capacity, a distinctive differentiation pattern could be described. At culture, inception cells from the lines were vimentin and laminin. Over time, several cytoskeletal and extracellular matrix (ECM) proteins indicative of vascular smooth muscle differentiation were expressed, including: the alpha-SM-actin isoform; the actin-binding proteins, smooth muscle myosin-heavy chain (SMMHC), SM1; h-caldesmon; calponin; gelsolin; and the ECM proteins, collagen IV and elastin. In full-grown lines, cells were similar to immature, intimal, vascular smooth muscle cells as found beneath the endothelium in adult aortas. Because of the coupling between proliferation and differentiation, the differentiation pattern seems to be under genetic control. However, since the coupling was not stringent during the whole lifespan of the lines, it is possible that cytokines are also involved, ensuring autocrine regulation of CDCL development.