Differentiation of TER-119⁺/4A5⁺ cells isolated from the spleens of phenylhydrazine (PHZ)-treated mice in serum-deprived cultures stimulated with either erythropoietin (EPO; 5 U/mL) or thrombopoietin (TPO; 100 ng/mL).

(A) Flow cytometric analysis of the expression of TER-119 and 4A5 in cells isolated by immunomagnetic selection as such (left panel) and after 24-48 hours of culture in the presence of either EPO (center panels) or TPO (right panels). Negative controls (not shown), analysis conditions, and cell gating are the same as in Figure 1A. The frequencies of the cells expressing TER-119 and/or 4A5 observed in individual experiments are shown in Table 4. (B) May-Grunwald Giemsa staining of Ter119⁺/4A5⁺ cells purified from the spleens of PHZ-treated mice as such (left panel) or cultured for 24-48 hours in the presence of either EPO (center panels) or TPO (right panels). The same cell preparations presented in Figure 2A (magnification ×400). (C) Semiquantitative RT-PCR analysis of the expression of erythroid (β-globin) and megakaryocytic (AchE and GpIIb) genes in TER-119⁺/4A5⁺ cells purified from the spleens of PHZ-treated mice at baseline (left) or cultured for 24 hours in the presence of EPO (center) or TPO (right). Actin complementary DNA (cDNA) was amplified to control for the total amount of cDNA used in each reaction. The same cell fractions are shown in the top right panels of Figure 3A. β-globin and actin were amplified for 18, 21, and 24 cycles and AchE and GpIIb for 27, 30, and 33 cycles (increasing numbers of cycles are indicated by a triangle on top of the panels). Similar results were obtained in 3 additional experiments.