Co-transduction of cDNAs for c-kit and steel factor into single CD34(+) cord blood cells further enhances the growth of erythroid and multipotential progenitors

Previous studies have demonstrated that the c-kit encoded tyrosine kinase r eceptor and its ligand, steel factor (SLF), are critical for normal blood c ell development. We have reported that transduction of the c-kif gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blo od (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-tra nsducing c-kif and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels o f CD34 and different levels of c-kit. Cells were then prestimulated with gr anulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL) -3, IL-6, erythropoietin (Epo) in the presence and absence of various conce ntrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, a nd then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kif and SLF genes significantl y enhanced colony formation compared with individual gene transduction, esp ecially by erythroid and multipotential progenitors that responded to stimu lation by added cytokines. Little or no growth was seen with the c-kit- and /or SLF-transduced cells without addition of cytokines. The degree of enhan cement effected by co-transduction inversely correlated with the degree of expression of c-kit protein before transduction. Optimal enhancing effects were noted in CD34(+++) kit(Lo/-) cells co-transduced with both c-kit and S LF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF m RNA expression in CD34(+++) cells and enzyme-linked immunoadsorbent assay ( ELISA) measurement of secreted SLF protein demonstrated that the transduced SLF cDNA was expressed and soluble SLF was released in medium cultured wit h SLP gene transduced MACS-separated CD34(+) cells in the presence, but not in the absence, of IL-3, GM-CSF, IL-6, and Epo. These results demonstrate the enhancement of the proliferation of growth factor responsive HPC that e xpress transduced c-kit and SLF genes.