SELF RENEWAL IN NORMAL & LEUKEMIC HEMOPOIETIC STEM CELLS

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It is proposed to characterize active factors operating in recently developed methods for the in vitro culture and activation of pluripotential hemopoietic cells and to transfer DNA's for GM- CSF and MultiCSF into such cultured stem cells or their controlling stromal cells to determine the role of dysregulated CSF production in the development and progression of myeloid leukemia. The biochemical nature of the factors able to control the maintenance and expansion of pluripotential hemopoietic cell numbers will be determined using three types of starting materials (a) medium from the cloned B.Ad stromal cell line, (b) normal organ conditioned media (thymus, marrow shaft), and (c) U5637 conditioned medium. Biochemical fractionation will be monitored by three assay systems using post5FU bone marrow cells and will determine whether more than one such factor exists. Where appropriate, FACSpurified pluripotential cells will be used with purified recombinant CSF's to establish whether the stem cell-active factors have proliferative, activating or differentiation-inducing actions. DNA's for murine GM-CSF and Multi-CSF (IL-3) will be introduced (a) into hemopoietic stemm cells cultured with stimulation by the above factors, sing retroviral vectors and microinjection, then the cells used to repopulate irradiated recipients, and (b) into all tissues of the body by the generation of transgenic mice. The hemopoietic tissues from these contrasting mice will be analyzed to determine whether dysregulated CSF synthesis alone is sufficient for myeloid leukemia development or whether additional radiationinduced translations or the insertion of an additional oncgene are necessary.