TRANSPLANT BIOLOGY, GENE TRANSFER, AND STEM CELL SOURCES

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DESCRIPTION (Adapted from the applicant's abstract) The long-term goal of this study is to determine the optimal source of hematopoietic stem cells (HSC) for transplantation, gene transfer, and prevention of graft versus host disease (GVHD). While the majority of transplants are carried out using bone marrow (BM) as the source of stem cells, there is increasing interest in the use of both chemotherapy and/or growth factor-mobilized peripheral blood progenitor/stem cells (PB) and umbilical cord blood (CB) as alternative stem cell sources. In the allogeneic setting PB would be preferable for the donor, and CB has many advantages, including the potential to create cryopreserved cord blood banks and the possible reduced immunological reactivity of CB lymphocytes with consequent decrease in GVHD. In the autologous setting CB and mobilized PB may contain increased numbers of cycling HSC and, therefore, constitute better targets for retrovirally mediated gene transfer. One of the major problems in transplanting both alternative sources of stem cells and advances in gene transfer efficiency to clinical use is the lack of optimal human stem cell assay systems. Aim 1 will compare the stem cell content and growth factor and stromal cell responsiveness of highly enriched primitive resting G0 cells from BM, CB, and PB, to test the hypothesis that CB and PB are enriched for these cells. This aim will be achieved by using novel means to enrich for these cells and by testing them in in vitro and xenogeneic in vivo culture systems, in an effort to optimize an assay for human long-term repopulating stem cells. Aim 2 will examine the capacity of stem cells from these different sources to be genetically modified by conventional retroviral vectors or novel vectors pseudotyped to express growth factor genes in place of the retroviral envelope gene. GVHD remains a source of major morbidity and mortality after allogeneic bone marrow transplantation (BMT). One can hypothesize that PB and CB differ from BM with respect to the function of their Th1 or Th2 T cell subsets that could account for the reduced GVHD observed in transplants with cells obtained from these sources, and Aim 3 will examine alternative human HSC sources for the number and function of the cells that are implicated in GVHD. In a murine GVHD model, preliminary data show that reduction of interferon-gamma (IFN- gamma) production by culturing donor T cells in interleukin 4 (IL-4) before BMT prevents this inflammatory cascade; G-CSF appears to have a similar effect to IL-4. Aim 3 will explore the hypothesis that treatment of murine donor cells with G-CSF prevents the inflammatory cytokine cascade of GVHD. By these means it is hoped that the rationale for selecting stem cells for transplantation and for gene transfer in different clinical contexts will be placed on a more secure scientific basis, and that by understanding and preventing GVHD it will eventually be possible to extend transplantation more successfully across HLA barriers.