Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-alpha and transforming growth factor-beta 1

The c-kit receptor and its ligand, steel factor (SLF), are critical for opt imal hematopoiesis. We evaluated effects of transducing cord blood (CB) pro genitor cells with a retrovirus encoding human c-kit cDNA, CD34(+) cells we re sorted as a population or as 1 cell/well for cells expressing high level s of CD34(+++) and different levels of c-kit ((++), (+), (Lo/-)), transduce d and then cultured in the presence of granulocyte-macrophage colony-stimul ating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-k it, but not with control (neo only), virus significantly increased colony f ormation, especially by erythroid and multipotential progenitors. The enhan cing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were n oted in CD34(+++) kit(Lo/-) cells transduced with c-kit. The stimulating ef fect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT -PCR) analysis of SLF mRNA expression in CD34(+) cells, and use of neutrali zing antibodies to SLF and/or c-kit suggested the presence of endogenous, a lthough probably very low level, expression of SLF by these progenitor cell s. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-pr and tumor necrosis factor-a. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our resul ts suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate t his effect. (C) 1999 by The American Society of Hematology.