Distinct myeloid progenitor–differentiation pathways identified through single-cell RNA sequencing

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Abstract

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (LinSca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs) (LinSca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre–granulocyte-macrophage progenitor (pre-GM) (LinSca-1c-Kit+CD41FcγRII/IIICD150CD105). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

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Figure 1: Gata1 expression identifies distinct myeloid progenitor subsets.
Figure 2: GE and GE+ progenitor cells have distinct myeloid lineage potential.
Figure 3: Identification of lineage potential associated with myeloid pathways expressing Gata1 or not.
Figure 4: Derivation of eosinophils from GE+ GMPs and of neutrophils from GE GMPs.
Figure 5: Segregation of granulocyte-monocyte potential before dissociation from the lymphoid and Mk-E lineages.
Figure 6: Gene expression in hematopoietic stem and progenitor populations.
Figure 7: Flt3 expression subcategorizes the GE pre-GM population.
Figure 8: In vivo myeloid reconstitution of GE and GE+ myeloid progenitor cells.

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References

  1. 1

    Koenderman, L., Buurman, W. & Daha, M.R. The innate immune response. Immunol. Lett. 162 2PB, 95–102 (2014).

  2. 2

    Orkin, S.H. & Zon, L.I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644 (2008).

  3. 3

    Metcalf, D. Lineage commitment and maturation in hematopoietic cells: the case for extrinsic regulation. Blood 92, 345–347, discussion 352 (1998).

  4. 4

    Sadrzadeh, H., Abdel-Wahab, O. & Fathi, A.T. Molecular alterations underlying eosinophilic and mast cell malignancies. Discov. Med. 12, 481–493 (2011).

  5. 5

    Manz, M.G. & Boettcher, S. Emergency granulopoiesis. Nat. Rev. Immunol. 14, 302–314 (2014).

  6. 6

    Pronk, C.J. et al. Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell 1, 428–442 (2007).

  7. 7

    Luc, S., Buza-Vidas, N. & Jacobsen, S.E. Delineating the cellular pathways of hematopoietic lineage commitment. Semin. Immunol. 20, 213–220 (2008).

  8. 8

    Iwasaki, H. & Akashi, K. Myeloid lineage commitment from the hematopoietic stem cell. Immunity 26, 726–740 (2007).

  9. 9

    Orkin, S.H. & Zon, L.I. SnapShot: hematopoiesis. Cell 132, 712 (2008).

  10. 10

    Akashi, K., Traver, D., Miyamoto, T. & Weissman, I.L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197 (2000).

  11. 11

    Arinobu, Y. et al. Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages. Cell Stem Cell 1, 416–427 (2007).

  12. 12

    Adolfsson, J. et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295–306 (2005).

  13. 13

    Iwasaki, H. et al. The order of expression of transcription factors directs hierarchical specification of hematopoietic lineages. Genes Dev. 20, 3010–3021 (2006).

  14. 14

    Fujiwara, Y., Browne, C.P., Cunniff, K., Goff, S.C. & Orkin, S.H. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc. Natl. Acad. Sci. USA 93, 12355–12358 (1996).

  15. 15

    Shivdasani, R.A., Fujiwara, Y., McDevitt, M.A. & Orkin, S.H. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 16, 3965–3973 (1997).

  16. 16

    Yu, C. et al. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J. Exp. Med. 195, 1387–1395 (2002).

  17. 17

    Migliaccio, A.R. et al. GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1low mouse mutant. J. Exp. Med. 197, 281–296 (2003).

  18. 18

    Nei, Y. et al. GATA-1 regulates the generation and function of basophils. Proc. Natl. Acad. Sci. USA 110, 18620–18625 (2013).

  19. 19

    Jasinski, M., Keller, P., Fujiwara, Y., Orkin, S.H. & Bessler, M. GATA1-Cre mediates Piga gene inactivation in the erythroid/megakaryocytic lineage and leads to circulating red cells with a partial deficiency in glycosyl phosphatidylinositol-linked proteins (paroxysmal nocturnal hemoglobinuria type II cells). Blood 98, 2248–2255 (2001).

  20. 20

    Drissen, R. et al. Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression. Blood 115, 3463–3471 (2010).

  21. 21

    Suzuki, M., Moriguchi, T., Ohneda, K. & Yamamoto, M. Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation. Mol. Cell. Biol. 29, 1163–1175 (2009).

  22. 22

    Iwasaki, H. et al. Identification of eosinophil lineage-committed progenitors in the murine bone marrow. J. Exp. Med. 201, 1891–1897 (2005).

  23. 23

    Kiel, M.J. et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005).

  24. 24

    Forsberg, E.C., Serwold, T., Kogan, S., Weissman, I.L. & Passegué, E. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors. Cell 126, 415–426 (2006).

  25. 25

    Luc, S. et al. Down-regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential. Blood 111, 3424–3434 (2008).

  26. 26

    Luc, S. et al. The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Nat. Immunol. 13, 412–419 (2012).

  27. 27

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

  28. 28

    Orkin, S.H. Diversification of haematopoietic stem cells to specific lineages. Nat. Rev. Genet. 1, 57–64 (2000).

  29. 29

    Mancini, E. et al. FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors. EMBO J. 31, 351–365 (2012).

  30. 30

    Keeshan, K. et al. Tribbles homolog 2 inactivates C/EBPα and causes acute myelogenous leukemia. Cancer Cell 10, 401–411 (2006).

  31. 31

    Naiki, T., Saijou, E., Miyaoka, Y., Sekine, K. & Miyajima, A. TRB2, a mouse Tribbles ortholog, suppresses adipocyte differentiation by inhibiting AKT and C/EBPβ. J. Biol. Chem. 282, 24075–24082 (2007).

  32. 32

    Querfurth, E. et al. Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors. Genes Dev. 14, 2515–2525 (2000).

  33. 33

    Cantor, A.B. et al. Antagonism of FOG-1 and GATA factors in fate choice for the mast cell lineage. J. Exp. Med. 205, 611–624 (2008).

  34. 34

    Walsh, J.C. et al. Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity 17, 665–676 (2002).

  35. 35

    Kurotaki, D. et al. Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation. Blood 121, 1839–1849 (2013).

  36. 36

    Hock, H. et al. Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation. Immunity 18, 109–120 (2003).

  37. 37

    Chi, A.W. et al. Identification of Flt3CD150 myeloid progenitors in adult mouse bone marrow that harbor T lymphoid developmental potential. Blood 118, 2723–2732 (2011).

  38. 38

    Ohneda, K. et al. Transcription factor GATA1 is dispensable for mast cell differentiation in adult mice. Mol. Cell. Biol. 34, 1812–1826 (2014).

  39. 39

    Kondo, M., Weissman, I.L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).

  40. 40

    Luc, S., Buza-Vidas, N. & Jacobsen, S.E. Biological and molecular evidence for existence of lymphoid-primed multipotent progenitors. Ann. NY Acad. Sci. 1106, 89–94 (2007).

  41. 41

    Arock, M., Schneider, E., Boissan, M., Tricottet, V. & Dy, M. Differentiation of human basophils: an overview of recent advances and pending questions. J. Leukoc. Biol. 71, 557–564 (2002).

  42. 42

    Vieira-de-Abreu, A., Campbell, R.A., Weyrich, A.S. & Zimmerman, G.A. Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum. Semin. Immunopathol. 34, 5–30 (2012).

  43. 43

    Nagasawa, T. et al. Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates. Front. Immunol. 5, 445 (2014).

  44. 44

    Abraham, S.N. & St John, A.L. Mast cell-orchestrated immunity to pathogens. Nat. Rev. Immunol. 10, 440–452 (2010).

  45. 45

    Cumano, A., Paige, C.J., Iscove, N.N. & Brady, G. Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356, 612–615 (1992).

  46. 46

    Lacaud, G., Carlsson, L. & Keller, G. Identification of a fetal hematopoietic precursor with B cell, T cell, and macrophage potential. Immunity 9, 827–838 (1998).

  47. 47

    Kawamoto, H., Ohmura, K. & Katsura, Y. Direct evidence for the commitment of hematopoietic stem cells to T, B and myeloid lineages in murine fetal liver. Int. Immunol. 9, 1011–1019 (1997).

  48. 48

    Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. Bipotential B-macrophage progenitors are present in adult bone marrow. Nat. Immunol. 2, 83–88 (2001).

  49. 49

    Görgens, A. et al. Revision of the human hematopoietic tree: granulocyte subtypes derive from distinct hematopoietic lineages. Cell Rep. 3, 1539–1552 (2013).

  50. 50

    Miyawaki, K. et al. CD41 marks the initial myelo-erythroid lineage specification in adult mouse hematopoiesis: redefinition of murine common myeloid progenitor. Stem Cells 33, 976–987 (2015).

  51. 51

    Moreira, P.N., Pozueta, J., Giraldo, P., Gutiérrez-Adán, A. & Montoliu, L. Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection. Methods Mol. Biol. 349, 151–161 (2006).

  52. 52

    McKenna, H.J. et al. Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 95, 3489–3497 (2000).

  53. 53

    Lindeboom, F. et al. A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse. Nucleic Acids Res. 31, 5405–5412 (2003).

  54. 54

    Kuhn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995).

  55. 55

    Kanakura, Y. et al. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors. Blood 71, 573–580 (1988).

  56. 56

    Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

  57. 57

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

  58. 58

    Roberts, A., Pimentel, H., Trapnell, C. & Pachter, L. Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27, 2325–2329 (2011).

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Acknowledgements

We thank A. Cumano (Pasteur Institute, Paris) for OP9 and OP9-DL1 stroma cells; the EMBL Monterotondo Gene Expression Service and Transgenic Core Facility for generating the Gata1-EGFP bacterial artificial chromosome and the corresponding transgenic mouse line, and flow cytometry facilities at the ISCR (supported by Wellcome Trust Equipment Grant WT087371MA (C.N.) and WIMM (supported by Medical Research Council Grants MC_UU_12009, MC_UU_12025 and G0902418) for flow cytometry and cell sorting. Supported by the Association for International Cancer Research (11-0724 to C.N.), the Medical Research Council (UK Program Grant G0801073 and Unit Grant MC_UU_12009/5 to S.E.W.J.; Strategic Grant G0701761, Program Grant G0900892 and Unit Grant MC_UU_12009/7 to C.N.) and the European Commission FP7 (CardioCell project (C.N.) and EuroSyStem project (S.E.W.J.)).

Author information

R.D., N.B.-V., P.W., A.M., E.S., S.E.W.J. and C.N. designed the experiments; R.D., N.B.-V., P.W., A.Ga., A.Gi., A.Z., M.L. and A.Gr. performed the experiments; S.T. performed gene-expression analysis; E.M. generated the Gata1-EGFP bacterial artificial chromosome transgenic mouse line; and R.D., S.E.W.J. and C.N. wrote the paper.

Correspondence to Sten Eirik W Jacobsen or Claus Nerlov.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations.

(a) Unsupervised clustering according to 100 top variable genes across single pre-GM cells. The two main cell clusters are indicated. (b) List of genes differentially expressed between clusters A and B in (a). Gene IDs, P-values and false discovery rate (FDR) q-values are indicated. Genes were included if both P<0.001 and q<0.05. (c) Structure of the Gata1-EGFP transgene (not to scale). The Gata1 genomic locus was contained within the RP23-443E19 BAC. (d-e) Flow cytometry profiles and gating strategies for identification of pre-GM, GMP, pre-Meg-E, MkP, pre-CFU-E and CFU-E progenitors (d) and LSKCD150+Flt3 and LSKFlt3hi cells (e).

Supplementary Figure 2 Defining HSCs and LMPPs based on Gata1.

(a-d) 22 weeks in vivo peripheral blood reconstitution after transplantation of 100 FACS purified GE or GE+ LSKCD150+CD48 cells together with 250,000 congenic whole BM cells. Values show reconstitution of the compartment analyzed (mean ±SD); n=3 recipients/population in one experiment. WBC; white blood cells. (e) For evaluation of megakaryocyte (Mk) potential, LSKFlt3hi GE and GE+ cells were manually plated at 1 cell per well into 60 well Terasaki plates and cultured for 8 or 12 days. Megakaryocytes were evaluated using an inverted microscope. Results are from 2 experiments, shown as percentage of colonies containing Mk cells of the total number of colonies formed. n=240 cells/population in 2 experiments. (f) Histogram plot showing expression of Flt3 and Gata1 in single sorted GE (left) and GE+ (right) pre-GMs. Expression is shown as -(Ct(gene)-Ct(Kit)). (g) Combinations of one GE pre-GM and three GE+ pre-GMs were cultured for 8 days. Single CD48HSCs were cultured for 25 days. Myeloid lineage potential readout of the cultures was based on morphology analysis after cytospins and MGG stain of the cultures. Number of cultures analyzed in indicated. Mo: monocytes, PMN: polymorphonuclear cells, Ma: mast cell. (h) Representative morphology of cells from cultures described in (g). Monocytes (Mo), polymorphonuclear granulocytes (PMN), and mast cells (Ma) are indicated. Scale bars: 25μm.

Supplementary Figure 3 Gene expression of vitro differentiated cell populations.

(a) Schematic overview of eosinophil favoring culture conditions used. (b) Morphology of peritoneal eosinophils (left panel) and peripheral blood neutrophils (right panel) purified by cell sorting after cytospin and May-Grünwald-Giemsa staining. Note the larger size and similar, but less condensed, nuclear morphology of eosinophils, compared to neutrophils. (c-f) Neutrophils (Ne), eosinophils (Eo), monocytes (Mo) and mast cells (Ma) from day 8 cultured pre-GM and GMPs as indicated were sorted based on markers as in Figure 4a. Their gene expression was analyzed by quantitative-PCR for genes associated with mast cells (c, red), neutrophils (d, yellow), eosinophils (e, purple) and Gata1/Gata2 (f, orange). Gene expression is presented relative to Hprt expression (mean ±SD) (n=2 biological replicates).

Supplementary Figure 4 In vitro differentiation of hematopoietic progenitors.

(a) Schematic overview of culture conditions used. Indicated progenitor populations were cultured for 3 days with mSCF and mIL-3, allowing the cells to reach a GMP stage, as defined by FcγRII/III expression. (b) Morphological analysis of EGFP-immunostained cytospins from cultures of the indicated in vitro generated EGFP and EGFP+ FcγRII/III+ cells after manually plating at a density of 5 cells/well and cultured for 8 days. Cell types are shown as a percentage of total number of cultures analyzed (numbers above bars). (c) Flow cytometry analysis demonstrate expression of CD55, Ly6C, integrin beta 7 (Itgb7) and interleukin 1 receptor-like 1 (Il1rl1) with Gata1-EGFP in pre-GMs and GMPs. Numbers indicate the percentage of cells within each quadrant. Representative plots of 2 (CD55, Ly6C and Il1rl1) or 1 experiment (Itgb7) are shown. (d) Quantification of identified cell population after 8-day cultures of CD55+ or Ly6C+ pre-GM and GMP by FACS as in Figure 4a. n=6-8 biological replicates. Error bars show SD. Mo: monocyte, Ne: neutrophil, Eo: eosinophil, Ma: mast cell. (e) Flow cytometry analysis of T-cell cultures (OP9-DL1 stroma) of LMPP and pre-GM GE cells cultured for 21 days (10 cells/culture). Plots show live cells. Stromal cells were gated out on FSC-SSC plot. Percentages are averages of cells in the gates or quadrants (+/− SD). (n=5 for LMPP, n=19 for pre-GM GE). Panels on the right show Thy1.2 vs CD25 expression profile on cells first gated as CD4CD8.

Supplementary Figure 5 Effect of absence of Flt3L and GATA-1 on myeloid progenitors.

(a) Flow cytometry analysis of wild type and Flt3L KO pre-GM cells from 17 week old male mice, showing Flt3 expression. Numbers indicate mean percentage of cells in each gate (+/− SD). n=5 from 2 experiments. (b) Quantification of number of myeloid progenitors by flow cytometry analysis shown as percentage of total bone marrow. Number of cells of indicated progenitors are shown as frequency of total number of bone marrow cells (+/− SD). n=5 from 2 experiments. (c) Schematic overview of Gata1 KO studies. Chimeric mice with wild type and conditional Gata1 KO blood cells are created by transplanting indicated cells into irradiated recipients. pIC is injected to delete the floxed Gata1 gene mediated by MxCre. GE and GE+ pre-GM and GMP cells were analyzed and sorted for in vitro culture. (d) Quantification of EGFP positive cells by flow cytometry of CD45.2 donor cells from experiment depicted in (c). Bars show percentage EGFP positive cells of the indicated CD45.2 progenitor population. Gata1fl/Y without MxCre are defined as wild type cells. n=2 for wild type cells and n=3 for Gata1 KO. SD is shown. (e) Indicated cells were manually plated at an average of 1 cell/culture and cultured for 8 days. Cell types of cultures are based on morphology analysis of cytospins. The number of cultures analyzed is indicated and are accumulated from 2 experiments. Mo: monocyte, PMN: polymorphonuclear granulocyte, Ma: mast cell (f) Representative picture of mast cells from cultured wild type and Gata1 KO GMP GE+ single cells. Scale bar is 25µM.

Supplementary Figure 6 In vivo myeloid reconstitution by GE and GE+ myeloid progenitor cells.

(a) Experimental design for direct comparison of in vivo potential from GE and GE+ GMPs and pre-GMs. (b-c) Quantification of in vivo contribution of GE+ and GE GMP (b) and pre-GM (c) progenitors to mast cells, eosinophils, neutrophils and monocytes, 7 (red; n=3) and 10 (blue; n=5) days after intra-femur transplantation of 2000-11000 cells. Mean (SD) donor derived cells per million analyzed MNCs per 1000 injected cells detected in injected femur (top) and spleen (bottom) is shown. Data is from three independent experiments. * below detection limit (see methods). (d) Total donor (CD45.2 + CD45.1/2) contribution per million MNCs analyzed at 7 and 10-11 days following IP or IF transplantation of indicated progenitor populations. Peritoneal fluid was analyzed from IP transplanted mice, and bone marrow cells from injected femur and spleen cells were analyzed from IF transplanted mice (n=3-5 mice). 1000-4000 cells/mouse were injected IP and 2000-11000 cells/mouse were injected IF. Data is adjusted to per 1000 injected donor cells per 1 million acquired MNCs. (e-h) Representative morphology of donor-derived monocytes (e), neutrophils (f), eosinophils (g) and mast cells (h) following IP transplantation of purified GMPs, pre-GMs and LMPPs. (i) Gata1-EGFP expression in donor-derived (CD45.1/2 and CD45.2) mast cells, eosinophils, neutrophils and monocytes 7 and 11 days following I.P. transplantation of GMPs and pre-GMs respectively. Data from transplanted mice included in Figure 8 is shown.

Supplementary Figure 7 Revised model of the hematopoietic hierarchy with an early bifurcation of distinct myeloid lineage pathways.

(a) The classical model with initial segregation of myeloid/Mk/E and lymphoid potential via a CMP and CLP, with a common CMP-derived GMP that gives rise to all monocyte-macrophage and granulocyte lineages. (b) Model incorporating the LMPP, where lymphoid and megakaryocyte-erythroid potentials separate early, but both the LMPP and CMP produce the same GMP with combined potential for all monocyte-macrophage and granulocyte lineages. (c) Model supported by the work described herein based on an early branching point generating LMPPs and erythroid-megakaryocyte primed multi-potent progenitor (EMkMPP), where the monocyte-macrophage and granulocyte lineages separate along with the lymphoid and megakaryocyte/erythroid potentials, according to their Gata1 expression, generating progenitors restricted to eosinophil-mast cell (and likely basophil) fate (EoMP), or to neutrophil or monocyte-macrophage fate (preNM, NMP). The GATA-1 and Flt3 expression domains are indicated. It should be noted that there is no direct evidence yet that the Flt3 fraction of preNMs derive from multi-potent progenitors (MPPs), so this pathways remains hypothetical. (d) Composite model of the work described herein that includes a CMP placed upstream of both the EMkMPP and the preNM, assuming it contains all monocyte-macrophage and granulocyte potentials at the single cell level, something yet to be determined. For all models it should be noted that other commitment pathways are possible, given the lineage potentials of the progenitors involved. For example, direct commitment of MPPs or another multi-potent progenitor to an Mk-E fate, circumventing the CMP and/or EMkMPP cannot be ruled out. In addition, these models do not incorporate all established findings in the hematopoietic hierarchy, such as the sustained myeloid programming of CLPs and downstream lymphoid progenitors.

Supplementary Figure 8 Quantification of transcriptome coverage of pre-GM single-cell RNA-seq.

(a) Quantification of the number of sequencing reads that were mapped to a unique genome position for each single pre-GM cell. (b) Percentage of total reads that were mapped to a unique genome position for each single pre-GM cell. (c) The total number of transcripts to which sequencing reads could be uniquely mapped for each single pre-GM cell, using a detection limit of 0.5 reads per million per kilobase of transcript. Cells where <2000 genes were detected were excluded from the analysis (sample 81).

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Supplementary Figures 1–8 (PDF 9415 kb)

Supplementary Table 1

Top 50 up-regulated and top 50 down-regulated genes in GE+GMP/GE– GMP comparison (XLSX 37 kb)

Supplementary Table 2

Antibodies used for flow cytometry (XLSX 39 kb)

Supplementary Table 3

TaqMan assays used for gene expression analysis (XLSX 34 kb)

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Drissen, R., Buza-Vidas, N., Woll, P. et al. Distinct myeloid progenitor–differentiation pathways identified through single-cell RNA sequencing. Nat Immunol 17, 666–676 (2016) doi:10.1038/ni.3412

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