Abstract
Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells1, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations2,3,4. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality5,6. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs7,8,9,10,11, a finding that is supported by studies of unperturbed haematopoiesis2,3,4,12 and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs7,8,9,10,13. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency11,14. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.
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Acknowledgements
We thank A. J. Mead, D. Atkinson, A. Giustacchini and N. Ashley for expert assistance with the Fluidigm array platform (WIMM Single Cell Core Facility is supported by the Medical Research Council (MRC) MHU (MC_UU_12009), the Oxford Single Cell Biology Consortium (MR/M00919X/1), the WT-ISSF (097813/Z/11/B#) and the WIMM Strategic Alliance awards G0902418 and MC_UU_12025); P. Sopp and S. A. Clark for expert flow cytometry technical support and cell-sorting services (WIMM FACS Core Facility is supported by the MRC HIU, MRC MHU (MC_UU_12009), NIHR Oxford BRC and the John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170) and by WIMM Strategic Alliance awards (G0902418 and MC_UU_12025)); the Biomedical Services at University of Oxford for animal technical support; the EMBL Monterotondo Gene Expression Service and Transgenic Core Facility for generating the Vwf-tdTomato BAC and the corresponding transgenic mouse line; N. Iscove for KitW41/W41 mice; A. Cumano for OP9-DL1 stromal cells; R. Drissen and S. Duarte for discussions and assistance with the preliminary phase of the studies; A. Hillen, B. Wu and T. Bouriez-Jones for technical assistance. This work was supported by Marie Curie Early Stage Researcher Fellowship (J.C.), the MRC UK (G0801073 and MC_UU_12009/5 to S.E.W.J. and G0701761, G0900892 and MC_UU_12009/7 to C.N.), the Swedish Research Council (S.E.W.J.), the Knut och Alice Wallenberg Foundation (WIRM; S.E.W.J.), the Tobias Foundation (S.E.W.J.), StratRegen KI (S.E.W.J.), Bloodwise (project grant 15006 to C.N.) and a BBSRC Project Grant (BB/M024350/1 to C.N.).
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S.E.W.J. and C.N. conceptualized the research, with input from A.S.-P. and J.C. S.E.W.J., C.N., J.C., Y.M., L.M.K. and P.S.W. designed the experiments and analysed the data. J.C. and Y.M. performed all experiments except fate mapping, with assistance from T.C.L., A. Ga. and A. Gr. (single-cell transplantations), L.M.K., R.N. and V.A. (peripheral blood reconstitution analysis), H.B. (blood and progenitor reconstitution analysis) and F.G. (CD229/CD41 analysis). L.M.K. performed fate-mapping experiments with assistance from K.H. and A.M.L. S.E.W.J., C.N. and J.C. wrote the manuscript, which was subsequently reviewed and approved by all authors.
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Extended data figures and tables
Extended Data Figure 1 Characterization of Vwf-tdTomato and Vwf-eGFP co-expression in PB and BM.
a–c, Flow cytometry analysis (one experiment) of Vwf-tdTomato and Vwf-eGFP co-expression in PB platelets and erythroid cells (a), in Kit-enriched BM LSK CD34−CD150+CD48− cells showing representative gating strategy used in single-cell transplantation sorts (b), and in myeloid and erythroid progenitors (c). d, FACS profile of BM LSK CD34−CD150+CD48− cells in Vwf-tdTomato/Gata1-eGFP mice (two experiments). For a–d, mean percentages of parent gates are shown in each plot, data are from three mice.
Extended Data Figure 2 Stable long-term lineage-restricted reconstitution in recipients of single Vwf-tdTomato+ LSK CD34−CD150+CD48− cells with wild-type BM support cells.
a, Reconstituted mice transplanted with single Vwf-tdTomato+ LSK CD34−CD150+CD48− cell and wild-type BM support. Data are from 58 transplanted mice over the course of seven experiments. No statistically significant difference in the frequency of reconstituted mice between KitW41/W41 (Fig. 1a) and wild-type support was observed (P?=?0.46). b, c, Analysis of platelet, erythroid-, myeloid-and B- and T-cell contribution 16–18wks post-transplantation in platelet-restricted (b) and PEM-restricted (c) mice (plots representative of >40 single cell transplantation experiments). d, Reconstitution kinetics. Data are from 3 platelet-restricted mice, 5 PEM-restricted mice, 6 PEMB-restricted mice and 11 multilineage mice. e, Distribution of lineage-restricted reconstitution patterns in mice in a. No statistically significant difference in the frequency of each pattern between mice transplanted with KitW41/W41 (Fig. 1d, n?=?109 mice) and wild-type support (n?=?25) was observed. Multilineage (Multi), P?=?0.662; PEMB-restricted, P?=?0.785; PEM-restricted, P?=?0.769; PE-restricted, P?=?1.0; platelet-restricted, P?=?1.0. f, Distribution of lineage bias within reconstitution patterns shown in e. Statistically significant differences in platelet-bias frequency between patterns are indicated above the bars (*P?=?0.015; ***P?=?0.0002). Statistically significant differences in bias for each pattern between mice transplanted with KitW41/W41 (Fig. 1e) and wild-type support are indicated within the bars (PEMB: *P?=?0.022, ***P?=?0.0007; Multi: L-bias (L-bi) *P?=?0.026, PEM-bias (PEM-bi) *P?=?0.044). g, Overall lineage bias distribution in e (wild-type support, n?=?25 mice) and Fig. 1e (KitW41/W41 support, n?=?109 mice). L-bias, *P?=?0.034; No bias, *P?=?0.028; PEM-bi, **P?=?0.004. For e–g, statistical comparisons performed using two-tailed Fisher’s exact test (95% confidence interval).
Extended Data Figure 3 Analysis of PB lineage-restricted reconstitution by single Vwf-tdTomato+ LSK CD34−CD150+CD48− cells and spleen lymphocyte reconstitution analysis in lineage-restricted reconstituted mice.
a–f, Flow cytometry analysis of PB platelet, erythroid, myeloid and B and T lymphocyte reconstitution, 16–18 weeks post-transplantation of a single Vwf-tdTomato+ LSK CD34−CD150+CD48− cell. Data are representative of >40 single-cell transplantation experiments. Platelet-restricted (a, b, two different representative mice), PE-restricted (c), PEM-restricted (d), PEMB-restricted (e) and multilineage (f) stably reconstituted mice. g, Flow cytometry analysis of myeloid and lymphoid reconstitution in spleens 23 weeks post-transplantation, corresponding to the platelet-restricted PB reconstitution pattern in b. h, Reconstitution of PB platelets and spleen lymphocytes 16–44 weeks post-transplantation in mice with platelet-restricted (n?=?8 mice), PEM-restricted (n?=?7) and multilineage (n?=?10) reconstitution. The frequency of positive mice and the mean reconstitution in positive mice is shown. Data are mean?±?s.e.m. from 8 (platelet-restricted), 7 (PEM-restricted) and 10 (multilineage) over the course of 13 experiments.
Extended Data Figure 4 Vwf-tdTomato co-expression with CD229 and CD41 in BM HSCs.
a, Vwf-tdTomato expression in LSK CD34−CD150highCD48− cells with different CD229 expression levels in two different mice (top and bottom). Data are representative of four experiments. Percentages of parent gate and MFI are shown in each plot. b, Expression of CD41 and Vwf-tdTomato in LSK CD34−CD150highCD48− cells with different levels of CD229 expression. Data are mean percentage of parent gate (±s.e.m.) from three mice in one experiment.
Extended Data Figure 5 HSPC reconstitution analysis in lineage-restricted mice transplanted with single Vwf-tdTomato+ LSK CD34−CD150+CD48− cells.
a–c, Flow cytometry reconstitution analysis of BM and thymus HSPCs in a representative platelet-restricted reconstituted mouse at 23 weeks post-transplantation (a), and representative multilineage (b) and PEM-restricted (c) reconstituted mice 16 weeks post-transplantation. Plots are representative of 12 HSPC analysis experiments.
Extended Data Figure 6 Multipotency of single reconstituting LSK CD34−CD150+CD48− cells with in vivo lineage-restricted output.
a, In vitro-derived granulocytes (top row) and monocytes/macrophages (bottom row) generated by donor-derived LSK cells sorted from single HSC-transplanted mice with long-term platelet-restricted reconstitution. Each column shows results for one mouse and the cytospins are representative of analysis of nine platelet-restricted mice. Scale bars, 50?μm. b, FACS analysis of granulocytes (CD11b+Ly6G+) and monocytes/macrophages (CD11b+Ly6G−) generated by 25–50 donor-derived LSK cells from three platelet-restricted mice. Data are representative of four mice analysed in three experiments. c, Gene expression heat map of GM cells generated in vitro. Data are mean Ct values per mouse group (normalized to mean Ct value of Hprt1 and B2m) from one (multilineage) or two (platelet-restricted) mice in one experiment in which four GM wells were seeded per mouse. Control data are from approximately 1,000 fresh PB leukocytes.
Extended Data Figure 7 Long-term persistence of platelet-restricted and platelet-biased reconstitution patterns by multipotent HSCs.
a, Reconstitution of HSPC hierarchy in BM and thymus (26 weeks post-transplantation) of two secondary recipients of cells from a donor mouse (44 weeks post-transplantation) with sustained platelet-restricted reconstitution (additional secondary recipients of the donor in Fig. 3d) Orange circles, all mice positive; grey circles, no positive mice. Frequency and reconstitution percentage (mean?±?s.e.m.) of positives shown as well as, where indicated, the percentage of cells expressing Vwf-tdTomato (Vwf+). b, Sorting strategy for secondary transplantation of Vwf-tdTomato+ HSCs from the mice in Fig. 3f (representative gating of mouse 2). Percentages of parent gates are shown in each plot.
Extended Data Figure 8 Reconstitution of PB mature lineages in mice transplanted with single LSK CD34−CD150+CD48− cells with different Vwf-tdTomato and CD150 expression levels.
a, Frequency of reconstituted mice transplanted with a single Vwf-tdTomato− HSC with low or high CD150 level. LSK CD34−CD150+CD48− cells were index sorted and CD150highVwf-tdTomato− cells were defined as having CD150 expression levels overlapping with CD150 expression in Vwf-tdTomatomid–high LSK CD34−CD150+CD48− cells (see Fig. 4a), and Vwf-tdTomato− CD150low cells were defined as having lower CD150 expression levels than LSK CD34−CD150+CD48−Vwf-tdTomatomid–high cells. Data are mean?+?s.e.m. from 57 (low CD150) and 77 (high CD150) mice over the course of 11 experiments. No statistically significant difference was observed, P?=?0.24. b, Distribution of restriction patterns generated by single Vwf-tdTomato−CD150low (n?=?18 mice) and CD150high (n?=?17 cells). Data are from 18 (Vwf-tdTomato−CD150low) and 17 (Vwf-tdTomato−CD150high) mice. No statistically significant differences were observed: LM, P?=?0.60; multi, P?=?0.40; PEMB, P?=?0.60. c, Distribution of lineage bias in e. No statistically significant differences were observed: no bias, P?=?0.23; PE-bias, P?=?0.49; L-bias, P?=?0.12. d, e, Flow cytometry analysis of PB platelet, erythroid-, myeloid- and B- and T-cell reconstitution 4 weeks (top panels) and 18 weeks (bottom panels) post-transplantation in L-restricted (b) and L-biased (c) reconstituted mice. Data are representative of 14 single-cell transplantation experiments of Vwf-tdTomato− HSCs. For a–c, statistical comparisons performed using two-tailed Fisher’s exact test (95% confidence interval).
Extended Data Figure 9 HSPC reconstitution analysis in L-restricted and L-biased reconstituted mice transplanted with a single LSK CD34−CD150+CD48− cell.
a, b, Flow cytometry analysis of PB platelet, erythroid-, myeloid-, B- and T-cell reconstitution, and HSPC hierarchy reconstitution, in L-restricted (a, representative of three experiments) and L-biased (b, representative of two experiments) reconstituted mice at 23 weeks post-transplantation. c, Reconstitution percentages of HSPCs 22–23 weeks post-transplantation in L-restricted and L-biased reconstitution patterns. Data are from 6 mice except for Pro-B, PreGM, GMP, MEP, CFU-E, MkP and L-biased cells that are from 3 mice. Orange circles, all mice positive; grey, no positive mice; pink circles, positive and negative mice. Frequency and reconstitution percentage (mean?±?s.e.m.) of positives shown as well as, where indicated, the percentage of cells expressing Vwf-tdTomato (Vwf+).
Extended Data Figure 10 Reconstitution analysis of secondary recipients of BM cells from single-cell transplanted mice with L-restricted and L-biased reconstitution.
a, b, Flow cytometry analysis of PB platelet, erythroid-, myeloid-, B- and T-cell reconstitution 8 weeks post-transplantation of secondary recipients of BM cells from primary mice with L-restricted (a, representative of 3 experiments) and L-biased (b, representative of 2 experiments) reconstitution generated by a single LSK CD34−CD150+CD48− cell. PB and BM HSPC reconstitution analysis of the primary recipients is shown in Extended Data Fig. 9a, b. c, Reconstitution percentages of HSPCs in L-biased primary recipient mice 21–29 weeks post-transplantation (n?=?3) and their secondary recipients 17 weeks post-transplantation (n?=?9, that is three secondary recipients per donor). Data are represented as in Extended Data Fig. 9c. d, Reconstitution kinetics of mice in Fig. 4h. Data for multilineage are from 6 and 2 mice for Vwf-tdTomato− and Vwf-tdTomatolow, respectively, and from 12 mice for Vwf-tdTomatomid and Vwf-tdTomatohigh. Data for platelet-restricted are from 1 mouse for Vwf-tdTomatolow and Vwf-tdTomatomid and from 8 mice for Vwf-tdTomatohigh.
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This file contains Supplementary Table 1 (Antibodies and viability dyes used in flow cytometry) and Supplementary Table 2 (TaqMan gene expression assays used in Fluidigm analysis of in vitro myeloid and lymphoid cultures). (PDF 195 kb)
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Carrelha, J., Meng, Y., Kettyle, L. et al. Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells. Nature 554, 106–111 (2018). https://doi.org/10.1038/nature25455
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DOI: https://doi.org/10.1038/nature25455
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