Suzanne L. Kirby, M.D., Ph.D.

Dissertation research performed under the direction of Stuart A. Bentley

ABSTRACT
Hematopoietic stem cell functions in vivo are dependent upon close-range interactions with their complex cellular and extracellular microenvironment. Proteoglycans are important, multifunctional components of the microenvironment which have been shown to have growth modulatory functions in a number of systems. To investigate the possible roles of proteoglycans in hematopoiesis, we sought first to characterize their production by stromal cells. Our results indicate that the cloned stromal cell lines examined produced proteoglycans which were similar to those produced by the cells found in the hematopoietic microenvironment: fibroblasts, endothelial cells, and smooth muscle cells.  The significance of the differences observed in proteoglycan synthesis between different stromal lines, however, was unclear. It was clear, though, that hydrocortisone is an essential component to successful in vitro long-term hematopoiesis. We therefore chose to examine next the effects of hydrocortisone supplementation on proteoglycan synthesis. We found that hydrocortisone virtually and uniformly abolished heparan sulfate proteoglycan synthesis by stromal cells and altered the pattern of synthesis of the chondroitin/dermatan sulfate species. This may have relevance as to the growth factor binding activity of proteoglycan species in the microenvironment that both we and others have demonstrated.  Finally, as others had demonstrated enhancement of hematopoiesis in vitro by xyloside-mediated perturbation of proteoglycan synthesis, we chose to further examine this phenomenon using stromal lines alone and in coculture with factor-dependent hematopoietic progenitors. We confirmed that xyloside supplementation produced the predictable effects of stimulating free dermatan sulfate glycosaminoglycan chain synthesis and had little effect on heparan sulfate synthesis. In contrast to reports by others showing a stimulatory effect of xyloside on hematopoiesis, we found an inhibitory effect both in cocultures with stromal cells and in colony assays that appeared to be a direct and nontoxic effect on the hematopoietic cells themselves.