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 receptor and its ligand, steel factor (SLF), are critical for normal blood cell development. We have reported that transduction of the c-kit gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blood (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-transducing c-kit and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels of CD34 and different levels of c-kit. Cells were then prestimulated with granulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL)-3, IL-6, erythropoietin (Epo) in the presence and absence of various concentrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, and then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kit and SLF genes significantly enhanced colony formation compared with individual gene transduction, especially by erythroid and multipotential progenitors that responded to stimulation 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 enhancement 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 SLF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF mRNA 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 with SLF 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 express transduced c-kit and SLF genes.
Lu
L
L
Department of Microbiology and Immunology, Indiana University School of Medicine, 1044 West Walnut Street, Room 302, Indianapolis, IN 46202-5254, USA. llu@iupui.edu
Dai
M S
MS
Ge
Y
Y
Wang
L S
LS
Braun
S E
SE
Wait
C L
CL
Griffith
D J
DJ
Heinrich
M C
MC
Broxmeyer
H E
HE
D018952:Antigens, CD34 / Q000097:blood*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D016207:Cytokines / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics*; D004357:Drug Synergism; D015672:Erythroid Precursor Cells / Q000166:cytology; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016133:Polymerase Chain Reaction; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D016662:Virus Integration
D018952:Antigens, CD34; D016207:Cytokines; D018076:DNA, Complementary; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit