Abstract
The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding—a common and life-threatening side effect of many cancer therapies—and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy1. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells2,3,4, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype5,6,7,8 remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development9. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.
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Accessions
ArrayExpress
Data deposits
The microarray data can be found in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) with the accession number E-MEXP-3935.
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Acknowledgements
The authors thank the Biomedical Services at Oxford University, B. Wu and H. Ferry for technical assistance, and M. José Fernandez Nestosa, S. Luc and N. Buza-Vidas for discussions and experimental contributions. This work was supported by the Medical Research Council, UK Grant H4RPLK0 (to S.E.W.J.) and an MRC Program Grant and Strategic Award (to C.N.), by the European Commission FP7 CardioCell (to C.N.) and EuroSyStem (to S.E.W.J.) projects, and by the Association for International Cancer Research (to C.N.). A.S.-P. was supported by a postdoctoral BEOI fellowship from the Spanish Ministry of Education and Science.
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Contributions
S.E.W.J., C.N. and A.S.-P. designed the project, analysed data and wrote the manuscript. A.S.-P., I.C.M. and C.T.J. organized, performed and analysed most experiments. P.S.W., T.C.L. and A.M. participated in experimental design, statistical analysis and performed/analysed experiments. S.Mo. and S.T. performed analysis of gene expression data. C.C. contributed with cloning of targeting construct for the BAC transgenic mouse model. T.B.J. helped with peripheral blood reconstitution analysis in transplantation studies. O.C. performed stem-cell transplantation experiments. M.L. performed in vitro megakaryocyte culture experiments and helped with transplantations. S.Ma. performed PCR analysis of endothelial and osteoblastic cells. L.S., J.C.A.G., R.F., H.B., A.Gr., A.Ga., J.C. and P.T. were involved in reconstitution analysis. D.A. helped with single-cell qPCR experiments. S.-A.C. performed cell sorting. All authors read and approved the final manuscript.
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Extended data figures and tables
Extended Data Figure 1 vWF–eGFP expression in endothelial cells and mature blood cell lineages.
a, vWF–eGFP targeting construct. b–e, eGFP expression in adult mice in peripheral blood (n = 1) T cells (CD4+ CD8+), B cells (CD19+) and myeloid cells (Mac1+ NK1.1−) (b); CD4+and CD8+ single-positive and CD4+ CD8+ double-positive thymocytes (n = 1) (c); platelets (n = 7) and erythrocytes (n = 7) (d); and bone marrow endothelial Ter119−CD45−CD31+ cells (n = 4) (e). Mean percentages of parental gates are shown. f, g, Endothelial (f) and osteoblastic (g) gene expression in FACS-sorted Ter119−CD45−CD31+ vWF+ endothelial cells (mean ± s.d., n = 3) and Ter119−CD45−CD31−ALCAM+ Sca-1− osteoblasts (mean ± s.d., n = 3). Hprt was used as a housekeeping gene.
Extended Data Figure 2 vWF–eGFP expression in adult bone marrow precursors and progenitors.
a, b, Gating strategy for adult bone marrow mature B cells (B220+ CD19+), T cells (CD3+) and myeloid cells (Gr-1+ Mac-1+) (a) for analysis of vWF–eGFP expression (n = 3) (b). c, vWF–eGFP expression in erythroid cells (I: CD71highTer119medium; II: CD71high Ter119high; III: CD71medium Ter119high; IV: CD71low Ter119high) (n = 3). d, vWF–eGFP expression in bone marrow progenitors (MKP, preCFU-E, preGM and preMegE in Vwf-eGFPtg/+) (n = 3). e, vWF–eGFP expression in adult LSK CD150−Flt3hi lymphoid-primed multipotent progenitors (n = 7).
Extended Data Figure 3 vWF–eGFP mice report Vwf expression.
a, Expression of Vwf messenger RNA relative to Hprt in sorted vWF–eGFP+ and vWF–eGFP− LSK CD150+ CD48− CD34− cells isolated from 8–10-week-old Vwf-eGFP reporter mice. Mean ± s.e.m. results from five biological replicates are shown. ***P < 0.0001 (Student’s t-test). b, qPCR analysis showing Vwf mRNA levels relative to Gapdh in FACS-sorted LSK CD150+ CD34−CD48− cells isolated from 8-week-old wild-type (WT; n = 2) and transgenic (TG; n = 3) Vwf-eGFP littermates. Each bar shows one biological replicate determination. Gapdh gene expression was used for normalization. c, d, Co-expression on HSCs of vWF–eGFP with CD49b and CD201 (n = 4) (c) and CD41 and CD86 (n = 3) (d). Mean frequencies of parental gate. e, Mean percentages of gated vWF+ and vWF− HSCs with side population (SP) phenotype (n = 2). f, Cell cycle analysis of vWF+ and vWF− HSCs and CD34+ multipotent progenitors (mean percentages, n = 2, pool of 7 mice each). DAPI, 4′,6-diamidino-2-phenylindole. g, Left, mean (± s.e.m.) total colony formation (white bars) and colonies containing megakaryocytes (grey bars) from single vWF–eGFP+ and vWF–eGFP− LSK CD150+ CD48− HSCs (n = 3). Right, typical multilineage colony with megakaryocyte and granulocyte/macrophage lineages.
Extended Data Figure 4 Reconstitution experiments.
a, Experimental outline to evaluate multi-lineage reconstitution and self-renewal potential of LSK CD34−CD150+ CD48− eGFP− and eGFP+ HSCs. vWF+ and vWF− HSCs (CD45.2), either single-cell-sorted by FACS-based automated cell deposition unit or 50 cells bulk sorted, were competitively transplanted along with 1 × 106 w41/w41 unfractionated bone marrow (BM) cells (CD45.1) or 2 × 105 wild-type unfractionated bone marrow cells (CD45.1). Cells were injected into lethally irradiated CD45.1 recipients. Peripheral blood (PB) reconstitution analysis was determined at indicated time points. The HSC compartment was analysed at 32 weeks after transplantation (TP) and bone marrow cells were retrieved from 50-cell recipients and re-transplanted into secondary hosts. b, Sorting of eGFP− and eGFP+ Lin−Sca-1+ c-Kit+ CD150+ CD48−CD34− cells was performed on c-Kit-enriched bone marrow cells and used for functional (competitive transplantation and hierarchical transplantation experiments) and molecular (global gene profiling and multiplex qPCR) assays. c, Mean (± s.e.m.) contribution of 50 vWF+ and vWF− LSK CD34−CD150+ CD48− HSCs to peripheral blood platelets, myeloid cells, B cells and T cells 12 weeks after secondary transplantation (n = 12 mice). **P < 0.01 (Student’s t-test).
Extended Data Figure 5 Gating strategy for peripheral blood reconstitution analysis.
a, b, White blood cell and platelet reconstitution analysis in mice 16 weeks after competitive transplantation of CD45.2 vWF+ HSCs (a) and vWF− HSCs (b). Donor platelets were identified based on scatter properties (SSC, side scatter) CD150, CD41 and eGFP expression as indicated. Peripheral blood leukocytes were gated as B cells (CD19+), T cells (CD4/CD8+) and myeloid cells (Mac1+ NK1.1−); and the percentage donor/test-cell (CD45.2) contribution to each lineage was calculated from these gates.
Extended Data Figure 6 Lineage-biased reconstitution patterns in mice competitively transplanted with limiting numbers of vWF− and vWF+ HSCs.
a, Gating strategy to analyse plasma-derived platelets (CD41+ CD150+) and myeloid (Mac1+ NK1.1−), B (CD19+) and T (CD4+ CD8+) cells in peripheral blood from competitively transplanted mice. b, Representative FACS plots showing minimal background staining of peripheral blood from CD45.1 mouse stained with anti-CD45.2 antibody. c, Representative FACS plots showing lymphoid-biased reconstitution pattern in peripheral blood of mice transplanted with 10 vWF− LSK CD34−CD150+CD48− cells at 16 weeks (mouse 1) or 27 weeks (mouse 2) after transplantation. Platelet or platelet/myeloid reconstitution patterns were not observed in any vWF− HSC recipients. Also shown are representative 16-week platelet-biased (mice 3–4) and lymphoid-biased (mouse 5) reconstitution patterns in mice transplanted with 10 vWF+ LSK CD34−CD150+ CD48− cells. Percentage test-cell (eGFP+ platelets or CD45.2+) contribution to the different lineages is indicated. d, Platelet/myeloid-biased peripheral blood reconstitution in mice transplanted with 5 vWF+ LSK CD34−CD150+ CD48− cells as analysed at 24 (mice 8 and 9) or 31–33 (mice 6 and 7) weeks after transplantation. e, FACS plots showing donor contribution to platelets (eGFP+) and white blood cells (CD45.2+) in mice reconstituted with a single vWF+ LSK CD34−CD150+CD48− HSC in Fig. 1l.
Extended Data Figure 7 Test-cell contribution to HSC and progenitor compartments in recipients of limiting numbers of vWF− and vWF+ HSCs.
a, Gating strategy to analyse HSC and progenitors in bone marrow. b, Representative FACS plots showing staining background in CD45.1 bone marrow stained with anti-CD45.2 antibody. c, Donor contribution to HSC compartment, eGFP expression in donor-derived HSCs and donor contribution to bone marrow progenitor compartments at 29 weeks in recipients, with identity numbers corresponding to peripheral blood analysis in Fig. 1. d, Donor contribution to HSC compartment, eGFP expression in donor-derived HSCs and donor contribution to bone marrow progenitors at 33 weeks in recipients of 5 vWF+ HSCs with identity numbers corresponding to peripheral blood analysis in Fig. 1.
Extended Data Figure 8 Acute depletion of platelets induces rapid cell cycle entry of platelet-primed LSK CD150+ CD48−vWF+ HSCs.
a, Outline of experimental design. b, Platelet levels in PBS and anti-CD42b-treated mice 24 h after treatment (n = 6). c, Plasma THPO levels in PBS (n = 2) and anti-CD42b-treated mice (n = 2) 24 h after treatment. Each bar shows one biological replicate determination. d, Representative cell cycle analysis of vWF− and vWF+ HSCs in control and anti-CD42b-treated mice 24 h after treatment. Percentages are mean values of 7 mice for control and 8 mice for anti-CD42b-treated mice, from three separate experiments.
Extended Data Figure 9 Expression of lineage-affiliated genes in HSCs in Thpo−/− mice.
a, qPCR analysis of expression of myeloid, erythroid and lymphoid genes in 9-week-old Thpo+/+ (black) and Thpo−/− (white) LSK CD34−Flt3− bone marrow cells relative to Hprt. Plots show mean (± s.e.m.) expression values of 6 replicate determinations. **P < 0.01, ***P < 0.001 (Student’s t-test). b, c, Mean contribution of transplanted Thpo−/−vWFtg/+ bone marrow cells (CD45.1) 18 weeks after transplantation to B-cell progenitors (n = 9) (b) and the HSC compartment (n = 4) (c).
Extended Data Figure 10 vWF+ and vWF− HSC contribution to B- and T-cell progenitors in secondary recipients.
a, Outline of experiments to establish hierarchical relationship between vWF–eGFP+ and vWF–eGFP− HSCs. b, Gating strategy for sorting of CD45.2+ Lin−vWF–eGFP− and CD45.2+Lin−vWF–eGFP+ cells for transplantation. Cells were sorted from primary recipients competitively transplanted with 150 vWF–eGFP−or vWF–eGFP+ CD45.2+ LSK CD150+ CD48−CD34− cells 6 weeks earlier and subsequently transplanted into secondary recipients in the hierarchical experiment outlined in a. Bottom panels show purity analysis for sorted populations; the percentage purity of each population is presented as the mean value from three individual experiments. c, Mean peripheral blood lineage contribution 16 weeks after secondary transplantation of CD45.2+ Lin−eGFP− or eGFP+ bone marrow cells. eGFP status of cells transplanted into primary and secondary recipients is indicated (n = 8 mice/group from 3 experiments). **P < 0.01, ***P < 0.001 (ANOVA). d–g, Gating strategies and test-cell contribution to CD4+ CD8+ thymocytes and B220+ CD19+ CD43+ bone marrow B-cell progenitors 18–20 weeks after secondary transplantation (n = 5) in hierarchical experiments. Representative plots from eGFP+/eGFP+ and eGFP−/eGFP− recipients are shown, including percentages of CD45.1 and CD45.2 contribution to each cell type. Reconstituted recipients/groups are indicated.
Supplementary information
Supplementary Table 1
This file contains configuration of FACS instruments. (XLS 21 kb)
Supplementary Table 2
This file contains antibodies, viability dyes and reagents used for FACS staining. (XLS 32 kb)
Supplementary Table 3
This file contains UPL assays used for Q-PCR analysis. (XLS 21 kb)
Supplementary Table 4
This file contains Taqman assays used for Q-PCR analysis. (XLS 25 kb)
Supplementary Table 5
This file contains custom prepared gene sets representing signatures of lineage restricted progenitors used for GSEA analysis. (XLSX 31 kb)
Supplementary Table 6
This file contains differentially regulated genes in Vwf- and Vwf+ HSCs by global gene expression profiling. Genes differentially expressed (adjusted P-value <0.05) between Vwf+ and Vwf– HSCs were identified using LIMMA. For these genes the Affymetrix probe set ID, gene symbol, P-value, adjusted P-value, and Vwf+/Vwf– ratio (log2 and linear scale) are shown. Normalized intensity values from individual arrays are also provided. (XLSX 65 kb)
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Sanjuan-Pla, A., Macaulay, I., Jensen, C. et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature 502, 232–236 (2013). https://doi.org/10.1038/nature12495
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DOI: https://doi.org/10.1038/nature12495
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