Single-Cell Transcriptome Analysis of Embryonic Erythro-Myeloid Progenitor Cells Reveals Lineage Heterogeneity

The paradigm that multipotential hematopoietic stem cells (HSC) differentiate through a hierarchical series of increasingly specified progenitors has recently been challenged by single cell transcriptome analyses, which suggest greater heterogeneity in progenitor populations and hierarchy than previously thought. These studies are complicated by the wide variety of hematopoietic progenitors at different stages of maturation concurrently present in the adult bone marrow. In the early mammalian embryo, unique hematopoietic progenitors arise prior to, and independently of, HSC. These include erythro-myeloid progenitors (EMP) that arise from hemogenic endothelium in the yolk sac and migrate to the fetal liver to jump-start definitive hematopoiesis before HSC emerge. In vitro, EMP can generate definitive erythroid, megakaryocyte and myeloid cells, although not B-cells, and include at least some single cells with both erythroid and myeloid potential (McGrath et al. 2015 Cell Rep. 11:1892). We have previously shown that day 9.5 (E9.5) murine EMP have a unique and uniform (kit+CD41+CD16/32+) immunophenotype in the yolk sac and circulation, which is maintained as they colonize the E10.5 liver. We performed single-cell transcriptome analysis of this apparently poised, embryonic-specific multi-potential hematopoietic progenitor population, asking whether it contains an underlying heterogeneity or intrinsic lineage hierarchy. High quality data, as assessed by transcriptomic complexity, read depth, and alignment distributions, were generated for 133 single-cell EMP (scEMP) derived from E9.5 mouse embryos. Use both of supervised and of semi-supervised analytical approaches uncovered transcriptome heterogeneity that allowed clustering of scEMP by expression patterns. Based on expression of known lineage-directing transcription factors and cell type enrichment analysis, clusters of cells were identified that could be annotated as multipotential hematopoietic, megakaryocyte/erythroid, myeloid, and a more specific macrophage lineage potential. A novel binary reduction data integration and transformation methods was developed and utilized to further refine and characterize single-cell classifications by facilitating cross platform comparisons with single cell RNA-seq data obtained from EMP progeny in E11.5 liver and with published gene expression datasets derived from adult hematopoietic progenitors and fetal macrophage populations. Predictions of lineage potential based on our analytical approaches were tested by sorting cells based on immunophenotypic markers differentially expressed in specific clusters and analyzing their potential using single cell colony-forming assays. These functional studies support the concept that immunophenotypically homogeneous EMP contain subpopulations of multipotential progenitors differentiating into erythro-megakaryocyte, myeloid, and macrophage-specific lineage fates.