The molecular characterization of bone marrow stromal cells and their role in hematopoiesis.

摘要

We have developed a unique series of murine stromal cell lines from a single long-term bone marrow culture. The lines support hematopoiesis to varying degrees. The primary focus of this study was to characterize, on a molecular level, the source of the supportive stromal phenotype. Initially, we screened the lines to determine their expression levels of a variety of known, hematopoietically relevant growth factors. Although the clones express a wide array of cytokines and growth factors, no particular pattern of growth factor expression could be correlated with either the supportive or non-supportive phenotype.; Upon further investigation, we found that differences in the cell lines' physiology were not limited to their hematopoietic supportive capacity. Under adipo-inductive culture conditions, certain clones exhibited widespread (morphological) differentiation into adipocytes. Under osteo-inductive conditions, several clones expressed dramatically increased levels of alkaline phosphatase, an enzyme associated with osteoblast activity. Using RNase protection assays, we discovered that many of the cell lines were expressing other osteogenic and mesenchymal factors. This data suggests that the lines possess a great deal of plasticity and may behave as mesenchymal stem cells.; Using representational difference analysis, or “subtractive hybridization,” we compared our most supportive line to our least supportive line in an attempt to uncover genes differentially expressed by the supportive clone. We found that the product of the growth arrest specific gene-6 (GAS-6) was differentially expressed by all of our supportive stromal lines. Transfection of GAS-6 into NIH3T3, a mesenchymal cell line that does not express GAS-6 and is incapable of long-term hematopoietic support, increased its supportive capacity 6–8 fold for up to six weeks in culture.; In situ immunostaining showed that GAS-6 transfected cells express a membrane-associated form of the protein while NIH3T3 and 3T3 transfected with empty plasmid do not. FACS analysis supported this observation and demonstrated that differences in fluorescence intensity were due specifically to surface-bound GAS-6. We propose that membrane-associated GAS-6 plays a critical role in mediating its activity in hematopoietic cultures.; Finally, we found that progenitor cells cultured on GAS-6 transfected cell lines demonstrate greater radioprotective ability when injected into lethally irradiated mice.