TrkA-RON activation can replace EPO in erythroblast expansion. (A) Erythroblasts undergo renewal division in the presence of EPO, SCF, and Dex, but they differentiate to mature erythroblasts in the presence of EPO. (B) Empty vector (EV) and TrkA-RON-transduced erythroblasts were seeded in medium containing no factors (○) or SCF and Dex supplemented with either EPO (2 U/mL; •) or NGF (10 ng/mL; □). Cells were counted daily, and cell densities were maintained between 1.5 and 3 × 106 cells/mL. Cumulative cell numbers were calculated. Error bars indicate SD of 3 independent experiments. (C) Cell morphology in the cultures described for panel B was examined on day 6. Cytospins were stained with histologic dyes and neutral benzidine to stain hemoglobin and were photographed with 60× magnification. Both cultures contained proliferating blasts in the presence of EPO/SCF/Dex. In the presence of NGF/SCF/Dex, control cultures (empty vector) contained pyknotic cells, whereas TrkA-RON-expressing cultures contained proliferating blasts. In the absence of factors, both cultures contained pyknotic cells. (D) TrkA-RON-expressing cells were factor deprived (-) and stimulated with NGF (100 ng/mL) or EPO (5 U/mL). EpoR was immunoprecipitated from cell lysates and was analyzed on Western blot with antiphosphotyrosine antibodies or specific antibodies to check for equal loading.
