CELL CYCLE INDUCTION OF CCE-27/c-kit POSITIVE CELLS BY HEMATOPOIETIC GROWTH FACTORS: EFFECTS ON GENE TRANSFER AND LONG TERM RECONSTITUTING ACTIVITY

Abstract

Hematopoietic growth factors (HGFs) and the bone marrow microenvironment act in concert to influence proliferation and differentiation events of a small pool of pluripotential primitive hematopoietic stem cells (PHSCs) in the ongoing process of hematopoiesis. The ability of PHSCs to self renew as well as differentiate into all known hematopoietic lineages is well documented; it is these qualities which make them attractive as targets for gene transfer. One of the major obstacles to the use of PHSCs for gene transfer has been the low frequency at which these cell divide, resulting in low frequency of infection by retroviral vectors. It is known that combinations of HGFs are able to induce proliferation in primitive cell populations, however, finding the balance between induction of cycling (to promote gene transfer) and maintenance of the pluripotential state (to promote long term expression of the transferred gene) has been difficult. The studies reported here were designed to evaluate the effects of in vitro culture in HGFs on short term and long term bone marrow reconstitution in lethally irradiated hosts as well as effects on maintenance of long term repopulating cells as measured by competitive repopulation, and effects on in vitro gene transfer efficiency. Unfractionated normal bone marrow (UBM) was separated by countercurrent centrifugal elutriation (CCE) at a flow rate of 27 ml/min (CCE-27) then further purified by fluorescence activated cell sorting (FACS) for cells expressing c-kit (CCE-27/c-kit) to isolate a population of primitive cells. Enrichment was confirmed by examining the ability of CCE-27/c-kit+ cells to proliferate in soft agar colony-forming assays in response to combinations of HGFs (IL-6+SF or IL-3+IL-6+SF) as compared with UBM or CCE-27 cells. Fluorescent labeling studies using PKH-26, a fluorescent integral membrane dye, demonstrated that CCE-27/c-kit cells could be induced to cycle by culture in IL-6+SF or IL-3+IL-6+SF. The inclusion of IL-3 however, elicits a significantly greater expansion of c-kit positive cells. The effect of cycle induction on short term reconstituting activity (STRC) and pluripotential long term reconstituting activity (LTRC) was determined by transplantation of C57B1/6, Ly 5.2 (donor) cells into C57B1/6, Ly 5.1 (host) lethally irradiated recipients. Culture for 3 days in the presence of IL-6+SF showed a 5 fold decrease in LTRC as compared with freshly isolated CCE-27/c-kit+ cells. Culture in IL3+IL-6+SF demonstrated a further decrease in LTRC activity as compared with fresh cells and cells cultured in IL-6+SF. These results were supported by results obtained from competitive repopulation studies which demonstrated that culture in IL-6+SF resulted in an 8 fold decrease in repopulating units as compared with fresh cells. The inclusion of IL-3 in the culture conditions for 3 days also had a significant impact on maintenance of LTRC activity, reducing repopulating units 13 fold in comparison to CCE-27/c-kit' cells cultured in IL-6+SF and 100 fold relative to freshly isolated CCE27/c-kit+ cells. Infection studies revealed IL-3+IL-6+SF is superior to the IL-6+SF combination of HGFs in promoting retroviral infection, achieving up to a 63% frequency of infection by co-culture with an irradiated viral producer (packaging) cell line. Taken together these results suggest that culture of CCE-27/c-kit+ cells for 3 days in the presence of IL-6+SF, and to a greater extent IL-3+IL-6+SF promotes significant induction of cell cycling thereby increasing the frequency of gene transfer by retrovirus. However, inclusion of IL-3 in the culture conditions reduces LTRC activity of this population of primitive cells to less than that observed in normal bone marrow making use of IL-3 as an agent to promote gene transfer into pluripotential stem cells ill-advised where maintenance of the pluripotential state is crucial.