Ex vivo fractionation of early progenitor cells for the purpose of removing leukemic cells presents difficulties in obtaining sufficient numbers of stem and progenitor cells for timely hematopoietic reconstitution. Since the number of potentially normal stem cells is only a fraction of those harvested, the fractionation procedures themselves often limit the number of stem and progenitor cells available for autotransplantation, thus prolonging the period of neutropenia following preparative therapy in the transplant patients. In this subproject, we will utilize a continuous perfusion ex vivo hematopoietic culture system to define the kinetics and dynamics of ex vivo hematopoietic expansion of early, selected hematopoietic cells from CML patients. Out primary goal will be to generate sufficient numbers of progenitor and LTCIC cells to promote rapid and stable engraftment. In addition, we will study the expression of bcr-abl mRNA in colonies derived from primary progenitor cells, and from the progenitor cells produced by maintained and expanded LTCIC cells in these perfusion cultures, to determine to what extent expression of this leukemic chimeric message is present in these cells. Similarly, we will evaluate the clonality of bcr-abl negative primary progenitors and LTCICs, to see whether a """"""""preleukemic"""""""" Ph- clone can be detected in these samples, and whether a clonal population disappears or emerges following perfusion culture. If the results of these studies are encouraging, for any one specific purging or selection technique, we will initiate a clinical trial in which selected cells will be expanded ex vivo in a clinical scale perfusion bioreactor system, and then reinfused into the patients following ablative chemotherapy. This approach will then be directly incorporated into the clinical autologous transplantation programs, as directed by Dr. Deisseroth.
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