American Society of Hematology

Figure 2

$Figure 2. Comparison of 3 different MEP purification strategies for enrichment of CFU-Mk/E. Human MPB mononuclear cells were fractionated using 3 different approaches and analyzed for colony formation in the dual Mk/E assay and methylcellulose as described. (A) Starting from previously gated Lin− cells, CD34+CD38+ cells (*) were equivalently selected using each of 3 MEP purification strategies. (B-D) The candidate MEP population (orange) is CD45RA− (x-axis) and either IL-3Ra− (CD123, strategy 1), FLT3− (CD135, strategy 2), or MPL+ (CD110, strategy 3). The corresponding CMP (blue) and GMP (purple) populations are highlighted for each strategy. CMPs were also sorted for comparison. Percentages of total CD34+Lin− cells appear with gates. (E) FMO control used for strict gating of PE negative (bottom left gate) vs positive events shown in B-D. (F) For the dual Mk/E assay, colonies were enumerated based on dual immunohistochemistry for CD41a and GlyA. (G) For methylcellulose, colonies were enumerated based on typical morphology and validated with Wright-Geimsa–stained cytospins of selected colonies (data not shown). Results presented as an average for each colony type + standard deviation (SD) for ≥4 independent experiments except for strategy 1 (n = 3 for dual Mk/E and n = 2 for methylcellulose assays).$

Comparison of 3 different MEP purification strategies for enrichment of CFU-Mk/E. Human MPB mononuclear cells were fractionated using 3 different approaches and analyzed for colony formation in the dual Mk/E assay and methylcellulose as described. (A) Starting from previously gated Lin⁻ cells, CD34⁺CD38⁺ cells (*) were equivalently selected using each of 3 MEP purification strategies. (B-D) The candidate MEP population (orange) is CD45RA⁻ (x-axis) and either IL-3Ra⁻ (CD123, strategy 1), FLT3⁻ (CD135, strategy 2), or MPL⁺ (CD110, strategy 3). The corresponding CMP (blue) and GMP (purple) populations are highlighted for each strategy. CMPs were also sorted for comparison. Percentages of total CD34⁺Lin⁻ cells appear with gates. (E) FMO control used for strict gating of PE negative (bottom left gate) vs positive events shown in B-D. (F) For the dual Mk/E assay, colonies were enumerated based on dual immunohistochemistry for CD41a and GlyA. (G) For methylcellulose, colonies were enumerated based on typical morphology and validated with Wright-Geimsa–stained cytospins of selected colonies (data not shown). Results presented as an average for each colony type + standard deviation (SD) for ≥4 independent experiments except for strategy 1 (n = 3 for dual Mk/E and n = 2 for methylcellulose assays).

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