Transduction of mut10 into human CD34⁺CB cells abrogates myeloid differentiation. CD34⁺ CB cells were infected with either FMEV-mut10 or control FMEV-GFP as described in “Materials and methods.” (A) Flow cytometric measurements of eGFP-positive cells and their mean fluorescence was determined for each cell population. The transduction efficiency for one experiment is indicated. (B) Western blot analysis confirmed the expression of a 30-kDa protein from both the human mut10 cDNA and the murine mutAatII cDNA from the FMEV vectors. Similar levels of mut10 C/EBPα protein were observed in CD34⁺ CB cells and murine Lin^neg BM cells (Figure 1D); the protein from FMEV-mut10 Phoenix cells serves as an internal control between blots, as this is the identical protein extract. The high levels observed in Phoenix cells are due to high copy numbers of the DNA transiently expressed in these cells. In contrast, the amount of protein in the transduced CD34⁺ CB cells and murine Lin^neg BM represents stable expression of an estimated 1 to 3 copies of the vector per cell. (C-F) Cytomorphology of human CD34⁺ CB cells infected with the control FMEV-eGFP (C,E) or FMEV-mut10 vector (D,F), sorted for eGFP expression, and cultured in GM-CSF, SCF, and IL3. Immediately after sorting (C-D), both cell cultures consisted of a minor fraction of erythroid progenitors (proerythroblasts and basophilic macroblasts) and a major fraction of myeloblasts and promyelocytes. The nuclear morphology of the mut10-positive cells (D) is obviously changed compared with the control (C). (E-F) In 14-day cell cultures, the control culture contains a wide spectrum of maturing erythroid and granulocytic cells, as well as macrophages (E), while the mut10-transduced hematopoietic cell culture consists predominantly of neutrophilic and eosinophilic promyelocytes (F). Giemsa staining. Original magnification, × 560.