Genomic approach to investigating ex vivo hematopoiesis

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DESCRIPTION (provided by applicant): High-dose chemotherapy with autologous or allogeneic stem-cell rescue results in prolonged pancytopenia that is accompanied by infectious and bleeding complications requiring antibiotic and transfusion therapy and, at times, prolonged hospitalization. Infusion of large numbers of ex vivo expanded hematopoietic cells -as a supplement to the conventional auto- or allograft - has the potential to close the window of neutropenia and/or thrombocytopenia. Furthermore, the recently discovered plasticity of hematopoietic stem cells suggests that these readily available stem cells may be used for generating autologous or allogeneic cells and tissues for non-hematopoietic cell and gene therapies. Success of such therapies depends on the ability to generate large numbers of cells with the desired, therapy-dependent state of cell differentiation. This remains an elusive task despite the great progress in basic and applied biology. Culture conditions, such as cytokine combinations and presentation, oxygen tension (pC2) and pH, alter stem- and progenitor-cell differentiation and proliferation with substantial patient-to-patient variability. Little is known about the underlying molecular biology of these effects, and specifically, about the large-scale transcriptional program during differentiation. Such knowledge has large predictive and diagnostic potential for both ex vivo and in vivo outcomes. Thus, a comprehensive examination of the transcriptional program of ex vivo expanded human primary myeloid cells - initiated with CD34+ cells - will be examined using 8,300-gene DNA microarrays, and key findings further explored using standard molecular-biology tools. Studies include examination of the temporal and differential transcriptional program of G and Mk cells cultured either under high or low pC2 and/or pH, and with different cytokine combinations. Specific issues to be examined include the extent to which apoptosis is linked to Mk differentiation; if Mk apoptosis employs a machinery similar to that of general apoptosis; and why, in contrast to Mk cultures, there is such a low level of apoptosis in G cultures. Furthermore, gone-clustering and regulatory-network techniques applied to DNA-microarray data may lead to the discovery of unknown Mk- and G-differentiation genes. These experiments will provide the basis for future studies in which clinical specimens could be examined in the context of clinical stem cell transplantation.