Research ArticleHEMATOPOIESIS

Identification of two distinct pathways of human myelopoiesis

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Science Immunology  24 May 2019:
Vol. 4, Issue 35, eaau7148
DOI: 10.1126/sciimmunol.aau7148
  • Fig. 1 Myeloid potential in predefined hematopoietic progenitor populations.

    (A and B) Gating strategy of populations in adult human bone marrow samples. Displayed are live, lineage-negative singlets, positive or negative for CD34 and CD38 as indicated above the plots. CD10 was included in the lineage cocktail. Data are representative of seven donors in 16 experiments, and the numbers show gated cells as percentage ± SD of the parental gate. PECy7, phycoerythrin cyanine 7. (C) Histogram showing cell type determined by morphology of cytospins from single-cell cultures of CMP (n = 181), LMPP (n = 75), and GMP (n = 77) as percentage of total number of cultures analyzed. Mo, monocyte; Ne, neutrophil; Eo, eosinophil; Ma/Ba, mast cell/basophil; Mixed, neutrophil/monocyte in combination with eosinophil/basophil/mast cell morphology. Data are summary of six independent experiments (table S1A). (D to G) Morphology of single-cell cultures from CMP (D and E), GMP (F), or LMPP (G), representative of the data in (C). Scale bars, 25 μm.

  • Fig. 2 Heterogeneity of human CMPs revealed by single-cell RNA sequencing.

    (A) Plot indicating the CD45RA and CD123 expression measures by flow cytometry of the CMP, GMP, and MEP cells used for single-cell RNA sequencing. FITC, fluorescein isothiocyanate. (B) Left: t-SNE plot of 237 CMP, 32 GMP, and 27 MEP cells showing seven clusters as indicated by coloring. The contour plot (red gradient color in the background) indicates the kernel smoothing density of cells. Right: The same t-SNE plot showing the individual cell types: CMP (gray), MEP (light blue), and GMP (dark blue). (C) GATA1, (D) KLF1, and (E) CEBPA expression [as log2 (CPM)] superimposed on the t-SNE plot from (B).

  • Fig. 3 Characterization of progenitor populations defined by CD114 and CD131.

    (A) Gene expression level of CSF2RB and CSF3R in single CMP cells in identified clusters. Y axis represents the expression level in the log2 (CPM) scale. X axis represents the cluster identification. Numbers below each plot show the number of expressing cells and the total number of cells within the respective cluster. Box plot shows median and quartile values, and whiskers show outlier values within 1.5 times of the interquartile range. (B) Plot indicating the CD114 and CD131 flow cytometry signals of index-sorted single CMP cells used for gene expression analysis. PE, phycoerythrin. (C) Heat map showing gene expression of index-sorted CD131+ and CD114+ CMPs (indicated on top) as shown in (B). The genes were chosen as markers for the indicated clusters (left). Cells were clustered manually, and the gene expression was used to annotate the cells to one of the clusters (bottom). (D) Flow cytometry plot showing CD114 and CD131 signals of CMP cells and gates used for sorting CMP subpopulations. Numbers are cells within each gate as average percentage ± SD of parental gate. Data are representative of 11 independent experiments using six different donors.

  • Fig. 4 Measurement of lineage output from CD114+and CD131+CMPs.

    (A) Gating strategy for flow cytometry analysis of bulk cultured progenitors. Examples of cultures of CD114+ CMPs (top) and CD131+ CMPs (bottom) are shown. Arrows in the top panel indicate sequential gating. APC, allophycocyanin. (B) Quantification of cell types identified as in (A) after culture of indicated progenitor populations, presented as frequency of total defined cells. Data are averaged from five (GMP) or six (CMP CD114+ and CMP CD131+) donors, and 2 to 10 cultures were grown for each population. (C) Number of live cells retrieved from cultures in (B) measured by flow cytometry. P values indicate significance of differences in cell numbers (Mann-Whitney U test).

  • Fig. 5 Lineage affiliation of prospectively isolated myeloid progenitors.

    (A) CMP cells were index-sorted and cultured under MME conditions. The CD131 and CD114 expression of cells with at least one positive lineage readout indicated. The data are cumulative from two independent experiments (table S1B). (B) Venn diagram of lineage output from cells in (A) with positive lineage readout defined by the presence of both appropriate morphology and lineage-specific gene expression. (C) CD114 and CD131 intensity of isolated CMPs that gave rise to cultures containing mast cells, basophils or eosinophils (orange), or monocytes or neutrophils (blue). (D) Venn diagram as in (B) showing the overlap between positive eosinophil, mast cell/basophil, megakaryocyte, and erythrocyte lineage readouts. (E and F) Cytospins of single CMP cultures showing (E) erythroid (Er) and megakaryocyte (Mk) morphology and (F) erythroid, megakaryocyte, and basophil (Ba) morphology. (G) Lymphoid potential of indicated progenitor cell populations (30 cells per culture) grown in B/NK cell conditions (MS-5 stromal cells) or T cell conditions (OP9-hDL1 stromal cells) shown as frequency of cultures producing B, NK, or T cells. Data are from two donors in two independent experiments. Numbers above bars indicate number of cultures with a positive readout/number of cultures.

  • Fig. 6 Gene expression in prospectively isolated progenitor populations.

    (A) The indicated cell populations were purified from four independent bone marrow donors and RNA-sequenced. The box-and-whisker plots show the expression of CSF2RB and CSF3R. Boxes show mean and central quartiles; whiskers show data range. Individual data points are overlaid on the plots. (B) The three-dimensional plot shows PCA using the first three components derived from 1868 genes [P < 0.05 (ANOVA) and CV ≥ 0.3]; encircled areas indicate clusters containing myelo-erythroid progenitor cells with or without GATA1 expression as indicated. (C to K) Gene expression levels measured by RNA sequencing of the indicated genes, shown as in (A).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaau7148/DC1

    Fig. S1. Candidate cell surface markers for separation of myeloid progenitor subsets.

    Fig. S2. Expression of CD131, CD114, and CD133 on hematopoietic stem and progenitor subsets.

    Fig. S3. Single-cell heat map of clustered CMPs.

    Fig. S4. Influence of culture conditions on lineage readout.

    Fig. S5. Gating strategy for isolation of human stem and progenitor cell subsets.

    Fig. S6. Myeloid and lymphoid potential of progenitor populations is reflected in their gene expression.

    Fig. S7. Proposed model of the human hematopoietic hierarchy.

    Fig. S8. Expression of CD11b on myeloid cell types generated in vitro.

    Table S1. Cloning efficiencies of myeloid progenitors.

    Table S2. Genes differentially expressed between CMP clusters.

    Table S3. Donor samples.

    Table S4. Antibodies used for flow cytometry and FACS.

    Table S5. Gene sets used for GSEA.

    Table S6. Cytokines used for progenitor assays.

    Table S7. TaqMan probes for qRT-PCR.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Candidate cell surface markers for separation of myeloid progenitor subsets.
    • Fig. S2. Expression of CD131, CD114, and CD133 on hematopoietic stem and progenitor subsets.
    • Fig. S3. Single-cell heat map of clustered CMPs.
    • Fig. S4. Influence of culture conditions on lineage readout.
    • Fig. S5. Gating strategy for isolation of human stem and progenitor cell subsets.
    • Fig. S6. Myeloid and lymphoid potential of progenitor populations is reflected in their gene expression.
    • Fig. S7. Proposed model of the human hematopoietic hierarchy.
    • Fig. S8. Expression of CD11b on myeloid cell types generated in vitro.
    • Table S1. Cloning efficiencies of myeloid progenitors.
    • Legend for table S2
    • Table S3. Donor samples.
    • Table S4. Antibodies used for flow cytometry and FACS.
    • Legend for table S5
    • Table S6. Cytokines used for progenitor assays.
    • Table S7. TaqMan probes for qRT-PCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Genes differentially expressed between CMP clusters.
    • Table S5 (Microsoft Excel format). Gene sets used for GSEA.

    Files in this Data Supplement:

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