Hematopoietic stem cells proliferate both in vivo and in vitro in close association with a heterogeneous group of adherent ceHs, termed the hematopoietic microenvironment. Long-term marrow cultures made up of adherent fibroblasts, endothelial cells, and adventitial cells effectively mimic the hematopoietic microenvironment and in murine long-term marrow cultures hematopoietic stem cells survive and proliferate in vitro for several months. However, hematopoiesis is sustained for shorter periods using human bone marrow. In contrast, culture of stem cells in vitro without an adherent cell population is associated with a rapid loss of reconstitution capacity, even in the presence of multiple hematopoietic growth factors. Thus, the proliferation of stem cells in long-term marrow cultures and engraftment of infused cells following bone marrow transplantation is dependent on direct stem cell-microenvironment contact. This evidence suggests that hematopoietic stem cell adhesion to the hematopoietic microenvironment may play a key role in control of stem cell proliferation. Little is currently known about the adherence of stem cells to cells making up the hematopoietic microenvironment. One obstacle to the molecular and biochemical analysis of the hematopoietic microenvironment is the complex nature of the adherent cell population. We propose to use recombinant retroviral vectors containing oncogenes to immortalize and clone multiple human bone marrow stromal cell lines derived from endothelial cells and fibroblasts to study in detail the adhesive interactions of hematopoietic stem cells with the normal cellular constituents of the hematopoietic microenvironment.
The aim of these studies is to determine the ligand/receptor interactions responsible for adhesion of stem cells to the microenvironment. These interactions are also likely to be important for seeding of the medullary cavity with hematopoietic stem cells following bone marrow transplantation. Such studies may therefore contribute new understanding about factors which regulate the movement of stem cells in and out of the medullary cavity. Using knowledge gained about stem cell-stromal cell adhesion, an additional focus of this work is to reconstitute an optimal microenvironment in vitro for the amplification of human stem cells without loss of the capacity to reconstitute hematopoiesis. These studies will concentrate on the use of a newly identified and cloned growth factor, stem cell factor (SCF), which stimulates primitive hematopoietic cells in vitro and corrects the bone marrow failure associated with the steel mutation in mice in vivo. Recent work suggests that the expression of this gene is important in the microenvironment regulation of stem cell proliferation. The goal of these studies is to generate a simple in vitro culture system which incorporates both stem cell adhesion and growth factor additions for the long-term survival of stem cells in culture. Finally, the optimal culture conditions delineated in these studies will then be used to support the selection and amplification of human hematopoietic stem cells into which the adenosine deaminase cDNA has been introduced by retroviral mediated gene transfer. Currently, a major obstacle to application of gene transfer methods successful in mice to larger animals, including humans, is the inability to effectively infect and select stem cells in vitro. This work has direct application to somatic gene therapy for the correction of the fatal genetic diseases of bone marrow derived cells.
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