Transduction of recombinant human erythropoietin receptor cDNA into daughter progenitors derived from single CD343+ cord blood cells changes the differentiation profile of daughter progenitors

Enhancement of proliferation and differentiation of erythroid progenitors by co-transduction of erythropoietin receptor and H-ras cDNAs into single CD343+ cord blood cells

“…CD34 3+ cells were obtained after sorting MACS separated CD34 + cells on a flow cytometer (Facstar plus , Becton Dickinson, San Jose, CA, USA) with FITC-conjugated anti-human CD34 antibody (anti-HPCA-2) or FITC-conjugated mouse IgG 1 antibody, as a control, purchased from Becton Dickinson Immunocytometry System (San Jose, CA, USA) as previously described. [13][14][15][16][17] The CD34 3+ cells constitute 20-30% of total CD34 + cells, which are enriched for stem cells and immature progenitor cells. 18 CD34 3+ cells were directly sorted as one cell/well into wells containing 0.1 ml semisolid culture medium.…”

“…The cells were cultured for 2 days, then transduced by adding viral supernatant at 20 l/well with 8 g/ml polybrene (Aldrich Chemical Company, Milwaukee, WI, USA) as described previously. [13][14][15][16][17]19 The cells remained in the same cultures for another 14 days for colony formation. It was noticed that 2 days after prestimulation, before addition of viral supernatant, fewer than 5% of the cells formed doublets and the predicted short halflife of the virus preparation suggests that Ͼ95% of wells have been exposed to gene transduction at single cell level.…”

“…Colony-forming unit (CFU)-GM, burst-forming unit (BFU)-erythroid (E), and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM)-derived colonies were scored under an inverted microscopy. [13][14][15][16][17][18][19] …”

“…[13][14][15][16][17]19 Two pairs of primers were used at 1 m in the PCR reaction. The primers and the PCR conditions for amplifying the transduced EpoR and H-Ras were used as described previously.…”

“…12 Our previous studies demonstrated that retrovirusmediated gene transduction of human (h) EpoR cDNA into single highly purified hematopoietic stem/progenitor cells or their single daughter progenitor cells from human umbilical cord blood (CB) resulted in increased numbers of detectable Epo-responsive erythroid progenitors. 13,14 This increase was further enhanced by co-transduction of both EpoR and the interleukin (IL)-9 receptor cDNAs. 15 We have also reported that transduction of H-Ras cDNA into the human erythroleukemic TF1 cell line and human CD34 + CB cells resulted in increased numbers of red cellcontaining colonies and increased expression of erythroidspecific gene expression associated with an induced premRNA of the hEpoR gene.…”