The overall purpose of this proposal is to define the in vitro interactions between human marrow stromal cells, hematopoietic stem cells, hematopoietic growth factors and extracellular matrix components in order to ' identify the optimal conditions that are necessary to initiate, expand and sustain hematopoiesis. The following specific aims will be pursued: 1. Examination of the role of metabolically active and inactive marrow stromal cells and growth factors in sustaining hematopoiesis in vitro. 2. Analysis of the interaction between human hematopoietic stem cells and components of the marrow extracellular matrix and definition of the effect of -matrix on the responsiveness of these cell populations to recombinant cytokines. It will be determined f the proliferative capacity and self-renewal capacity of purified human stem cell populations can be enhanced by adhesion to whole matrix, individual matrix components or mixture of such components. Special emphasis will be placed on establishing whether matrix-cytokine complexes will optimize in vitro stem cell self-renewal. These studies ill allow one to approach the in vitro reconstitution of relevant locales within the :arrow microenvironment that favor stem cell self-renewal and expansion. 3. In vitro reconstitution of the hematopoietic microenvironment by establishment of long term bone marrow cultures composed of purified matrix components, recombinant cytokines and purified marrow stem cell populations. An important aspect of these investigations hill be to determine if the purified stem cell populations can be expanded in numbers and heir full self-renewal and differentiation potential maintained. These efforts will result in new knowledge of the complex interactions between human stem cells, growth factors and components of the extracellular matrix. Such work will p provide the basis by which stem cell populations will be expanded in vitro and be used as rafts for clinical transplantation. Use of such """"""""stem cell transplants"""""""" would provide source of marrow that would be devoid of tumor cells and therefore be more suitable for autologous transplantation. Such stem cell transplants would also facilitate dosing of adequate numbers of stem cells that would potentially favor sustained engraftment following allogeneic marrow transplantation. GRANT=R01AI29976 Polyclonal activation of resting B lymphocytes by antigen receptor crosslinking with antiimmunoglobulin efficiently produces B lymphoblasts that are, dependent on further mitogenic stimulation to remain viable or to transit the cell cycle. In previous work we identified stimuli that maintain viability (phorbol esters and IL-4) or cause progression through the cell cycle (LPS, anti-Ig, phorbol esters + ionomycin, and undefined T cell derived lymphokines). While much is known about the effects of anti-Ig on resting B cells, relatively little attention has been paid to mechanisms by which B lymphoblasts progress through the cell cycle. The goal of this project will be to identify and further characterize the mechanism of action of stimuli that maintain B lymphoblast cell cycle progression. The role of Ca++ and protein kinase C in anti-Ig mediated activation of B lymphoblasts will be explored and compared to requirements for activation of resting B cells. A number of cytokines will be evaluated, alone and in combination, for ability to maintain viability of B lymphoblasts, and to synergize with stimuli already defined. B lymphoblasts obtained by other means, such as direct contact with T cells, will be evaluated for responsiveness to this same battery of agents. The expression of mRNA for hormone receptors and proto-oncongenes will be examined as a function of both B cell activation state and of the mode of activation. Finally, as we have found that expression of c-myb is cell cycle regulated in B lymphomas, the expression and role of this proto-oncogene in normal B cells, and its regulation by growth promoting stimuli will be determined. These studies should provide valuable information regarding intra- and extracellular signals that regulate B lymphoblast cell cycle progression.
