Investigation of the molecular and intra-molecular mechanisms, determining the cell differentiation direction in appropriate incubation conditions, on cellular and organism levels

The main goal of the current study was directed to derivation of one or another type of normal mature cells from partially-differentiated cells, by incubation in different laboratory conditions, but also to reveal the molecular mechanisms, underlining these maturation variations. In short-term (2-3 days) pre-cultivation of mouse embryonic 3T3 fibroblasts, in cultural fluid, in which mouse malignant myeloma cells, signs of early myeloid differentiation were noted. After freezing in the presence of the cryo-protector Dymethylsulfoxide (DMSO), subsequent thawing and in vitro-re-incubation, appearance of separated sub-populations of multi-nuclear osteoclast-like cells was observed. When the cultural fluids from the so derived osteoclast-like cells, were added to de novo-formed confluent monolayers of 3T3 embryonic cells, signs osteoblast-like cell differentiation was noticed, as appearance of dark-staining mineral depositions. In co-cultivation of cells from both cell types derived, zones of destructed osteoblast-like cell monolayer were seen. These data could be explained with eventual existence of separated stem/progenitor cells in the general 3T3 cell line, which are able to differentiate in respective directions depending of the respective incubation conditions. On the other hand, the role of 3T3 embryonic fibroblasts as feeder cells, supporting the proliferation of stem/progenitor cells from both human ocular cornea limbus and oral mucosa, was also indicated, which supported the literature findings about the importance of embryonic mouse fibroblasts as feeder cells, supporting stem cell proliferation, for the needs of clinical practice. This application of the embryonic mouse fibroblasts was proved for embryonic stem cells in our previous studies. Because epithelial stem/progenitor cells from human oral mucosa were proved to express limbal epithelial stem cell markers, they were in vitro-cultivated in similar laboratory conditions as limbal epithelial stem/progenitor cells. These results were in agreement with respective literature data, and could be explained with eventual existence of separated stem/progenitor cell sub-populations, which are able to differentiate in respective directions in appropriate cultivation conditions. In this way, any role of the extra-cellular matrix components on the cell differentiation activation and direction, as well as the influence of DMSO and other organic detergents on the processes of intra-cellular fusion in the formation of osteoclast-like cells, could be suggested. For study of the underlying events, which determine the differentiation of cell progenitors to one or another direction in respective incubation conditions, investigations on the direct and/or indirect intra-molecular (protein-protein, protein-DNA, protein-RNA, etc.) interactions, was necessary.