Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways

Abstract

BackgroundRecent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.ResultsTo clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: “Pre-MEP,” enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; “E-MEP,” strongly biased towards erythroid differentiation; and “MK-MEP,” a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output.ConclusionsOur study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0939-7) contains supplementary material, which is available to authorized users.

CRISPR/Cas9 genome editing in human hematopoietic stem cells

“…Representative FACS plots of subpopulations are depicted in Supplementary Figure 2 as well as in Figure 5f in Reinisch et al 52 , Figure 1 in Majeti et al 27 , and Figure 2a in Psaila et al 53 . Add the in vitro HSC antibody cocktail, mix well, and incubate on ice in the dark for 30 min.…”

Characterization, regulation, and targeting of erythroid progenitors in normal and disordered human erythropoiesis

“…These cells undertake a “myeloid bypass” whereby oligo- and bi-potent progenitors with self-renewing capacity at the single cell level give rise to erythroid, megakaryocyte and granulocyte-macrophage lineages (7). There is also growing evidence that the bipotent MEP contains multiple populations with varying lineage biases (8*, 9*). …”

“…As summarized in Table 1, MEPs have been characterized using a combination of cell surface markers including IL-3R, FLT3, MPL (assessed using BAH1 clone and other antibodies), CD36, CD41, CD71 and CD105 (8*, 9*, 10**, 14**). Using a combination of approaches including functional, single cell transcriptomics and lineage potential assays, these studies reveal that the identification of MEP and their subpopulations appears highly dependent on gating strategy; this likely reflects their extensive heterogeneity.…”

Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML

“…[17][18][19][20][21][22][23] In leukemia, single-cell methods additionally offer the opportunity to discriminate between leukemic and healthy cells, thereby allowing for specific characterization of the infrequent residual LSC population even months into treatment. Here we have dissected the heterogeneity of the CML LSC population both at diagnosis and following 3 months of TKI treatment.…”