Fig. 6
Single-cell functional assays confirm erythroid and megakaryocyte differentiation bias of CD71 + 41- MEP and CD71 + 41+ MEP, whereas CD44hi71- 41- MEP demonstrate a “Pre-MEP” phenotype. a Colony-forming capacity of single MEP cells in methylcellulose, which primarily supports erythroid and myeloid differentiation. Left graph: colony phenotype as a percentage of total colonies grown. The percentage of erythroid colonies (BFU-E/CFU-E; red) was significantly higher for CD71 + 41- MEP than the other two populations. CD44hiCD71-41- MEP cells generated a higher percentage of myeloid colonies (CFU-GEMM/GM; blue) than the CD71+ fractions. Photographs show representative BFU-Es derived from single CD71 + 41- and CD71 + 41+ cells. Data shown are for 30–60 single cells sorted from each population in each of seven separate experiments. b Megakaryocyte colony-forming potential was tested in a collagen-based assay supporting megakaryocyte and myeloid but not erythroid colonies. CD71 + 41+ MEP cells gave rise to significantly more megakaryocyte colonies (green; n = 4). c Typical cell cultures 6 days after seeding of single cells from MEP subsets into a liquid culture system supporting erythroid and megakaryocytic differentiation. An example of a mixed megakaryocyte and erythroid colony is shown for CD71 + 41+ MEP, with two large, proplatelet-forming megakaryocytes (green stars) and several smaller erythroblasts (red arrow). The example colonies shown for CD44hiCD71- CD41- and CD71 + CD41- MEP are exclusively erythroid, with a higher proliferation rate in the CD71 + 41- colony. d The identity of cells in individual culture wells was determined by immunofluorescence (IF) microscopy, flow cytometry, and morphology. Example IF images of mixed (Mix, top), megakaryocyte-only (MK, middle), and erythroid-only (Ery, bottom) cultures. Cells immunostained for CD71 (FITC, green) and CD41(APC, red). e Cell number in each well 6 days after seeding with a single cell. CD71 + 41- MEP are most proliferative. N = 3. f Summary FACS data for 100 single-cell colonies analyzed (n = 3). CD44hiCD71- 41-MEP most frequently generated mixed erythroid/megakaryocyte colonies; CD71 + 41- showed mostly erythroid-only and CD71 + 41+ showed primarily MK-only progeny. P values are for one-way ANOVA between populations