Fig. 2

Single-cell gene expression analysis demonstrates significant cellular heterogeneity and the presence of subpopulations within classically defined, immunophenotypic MEP. a A previously validated strategy was used to distinguish MEP from the other lineage-negative (Lin-) CD34+ CD38+ myeloid progenitor populations—common myeloid (CMP) and granulocyte-macrophage progenitors (GMP)—by the absence of CD123 and CD45RA. Quantification gates are shown (sorting gates are shown in Additional file 1: Figure S1A). b Multiplex qPCR of 87 genes in 489 Lin- CD34 + CD38 + CD123- CD45RA-MEP cells and PCA was performed. The distribution of cells along PC 1 demonstrates two distinct cellular populations (annotated 1 and 2). c Plot showing % variance by PCs 1–10. d Superimposition of mean log₂ fluorescence intensity (MFI) values of the original cells isolated for qPCR on the PCA for PC1 and PC2 reveals that the two populations have distinct expression profiles for CD34, CD38, and CD71. e Superimposition of CD41 and CD42 expression on the PCA for PC1 vs. PC2 (MFI, left plots) indicated rare cells with high CD41 and CD42 expression which did not fall into either Population 1 or 2, suggesting the presence of smaller subpopulation(s) expressing megakaryocyte-associated antigens. CD41^high and CD42^high cells segregated more distinctly by PC3 vs. PC4 (relative mRNA expression, right plots). Red-blue scale indicates high to low expression (customized for each plot in 2D and 2E). f Representative flow plot (left) illustrating differential expression of CD71 and CD41 within immunophenotypic MEP compartment, identifying three subpopulations: (1) CD71-CD41-; (2) CD71 + 41- ; and (3) CD71 + 41+. Quantification of these three subpopulations (right) in CD34+ cells from 14 healthy donors. Cells falling between FACS gates are excluded from the chart. CD71 + 41 + MEP are significantly less frequent, constituting 5.1 ± 0.6 % of total MEP (mean ± SEM, P <0.0001). g Expression of CD42 in the three MEP subfractions. CD42 expression is restricted to a minority (20.7 ± 4.1 %) of CD71 + CD41 + MEP cells (P <0.0001)