Human hematopoietic stem cells in marrow, adult peripheral blood and cord blood will be isolated by a combination of steps involving SBA agglutination, immunocytoadherence and immunomagnetic bead positive and negative selection for CD3+ , CD33- LIN- HLA-DR- cells. Additional separation by biophysical criteria, predominantly density, and immunofluorescence and rhodamine 123 fluorescence intensity by FACS analysis will be undertaken. Stem cell identification will be undertaken using an in vitro self-renewal (Delta) assay, a high proliferative potential (HPP-CFU) colony assay, a blast colony assay in methylcellulose or on marrow stroma, and a micro-assay based upon prolonged reconstitution of CFU and differentiated cell production on irradiated marrow stroma. Selective survival, cycle activation, self renewal and differentiation of early stem cells will be facilitated using combinations of cytokines with proven action at the stem cell level - IL-1, IL-3 and IL-6 .in combination with each other and with G-CSF, M-CSF and GM-CSF. The action of the c-kit protooncogene ligand (KL) on-stem cell proliferation will be explored, as will the stimulatory potential of an IL-3-GM-CSF hybrid molecule. The introduction of cytokine CSF genes by retroviral infection of appropriate marrow stromal cell lines will be used to develop long term culture systems for maintaining long term reconstituting stem cells. Lines will be selected for sustained production of, variously, G-CSF, GM-CSF, IL-1, IL-6, IL-3, and KL and retention of the ability to selectively cytoadhere early stem cell populations as determined by biological panning. Potential negative regulators of stem cell proliferation eg TGFbeta-1, MIP-la will be neutralized by appropriate antibodies in the various in vitro assay systems. The final criteria of stemness - long term, self-renewal and retention of pluripotentiality will be assessed on irradiated stromal cells by sustained hematopoiesis with maintenance of cells with a high delta value for prolonged periods. In vivo, stem cells will be inoculated into SCID mice bearing engrafted marrow stroma and establishment of human hematopoiesis within the bone ossicles will be monitored.
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