Myeloid Progenitor Cluster Formation Drives Emergency and Leukaemic Myelopoiesis Aurélie Hérault1*, Mikhail Binnewies1*, Stephanie Leong1*, Fernando J
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ARTICLE doi:10.1038/nature21693 Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis Aurélie Hérault1*, Mikhail Binnewies1*, Stephanie Leong1*, Fernando J. Calero-Nieto2, Si Yi Zhang1, Yoon-A Kang1, Xiaonan Wang2, Eric M. Pietras1, S. Haihua Chu3, Keegan Barry-Holson1, Scott Armstrong3, Berthold Gttgens2 & Emmanuelle Passegué1† Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1β, G-CSF, TGFβ and CXCL4) and activation of an inducible Irf8 and β-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia. Our understanding of blood production has evolved considerably over scattered throughout the BM cavity with no particular distribution the past years, mainly due to the introduction of new technologies to in relation to the bone endosteum, trabecular regions or central study haematopoietic stem cell (HSC) biology both in situ in their bone marrow, and were usually identified as individual cells intermingled marrow (BM) niche and at the clonal level. Recent tracking approaches with other haematopoietic populations (Fig. 1a). By contrast, in in native conditions, as opposed to transplantation experiments, have myeloproliferative neoplasm (MPN) models such as Scl-tTA::TRE- uncovered a prominent role for multipotent progenitors (MPPs) in BCR/ABL (BA)9 and junBflox/flox::MORE-cre (jB)10 mice, most GMPs maintaining blood production1,2. Detailed functional studies of the were found as part of large, compact clusters surrounded by lineage- MPP compartment have identified three distinct MPP subsets (MPP2, positive cells (Fig. 1b, Extended Data Fig. 1b, c). This process was MPP3 and MPP4) with unique lineage preferences that work together progressive and directly associated with disease development, starting to adjust blood production to the needs of the organism3,4. In particular, with loose GMP patches (pGMP) that evolved into large, compact increased production of myeloid-biased MPP2 and MPP3 by HSCs and GMP clusters (cGMP) (Extended Data Fig. 1d). We defined pGMPs myeloid reprogramming of lymphoid-biased MPP4 are now emerging as the collection of more than 4 GMPs loosely associated together, as key first steps of regenerative myelopoiesis4. which could also be occasionally observed at steady state in wild-type Myelopoiesis has long been considered to progress with the orderly mice, and distinguished them from cGMPs by the fact that they differentiation of MPPs into uncommitted common myeloid progenitors, were not tightly surrounded by mature lineage cells (Extended Data which then produce lineage-committed granulocyte/macrophage Fig. 1e). While GMPs were never found in wild-type spleens, we progenitors (GMPs) and megakaryocyte/erythrocyte progenitors5. observed abundant cGMPs in the disorganized red pulp of BA and Of note, single-cell RNA sequencing (RNA-seq) approaches6,7 and jB mice (Extended Data Fig. 2a). Similar features were observed in more refined barcoding lineage-tracking experiments8 indicate the MLL-AF9 (MF9) transplanted model of acute myeloid leukaemia pre-existing lineage specifications in common myeloid progenitors, (AML)11, with structures ranging from pGMPs to cGMPs (Extended and a large degree of molecular and functional heterogeneity in all Data Fig. 2b). Using a simplified version of this immunofluorescence myeloid progenitor populations. However, our knowledge of myeloid scheme, we investigated whether cGMP formation was associated with progenitor biology still lacks a precise understanding of how myeloid a major re-organization of the BM microenvironment, but found no differentiation occurs spatially in the BM cavity. Here, we used imaging obvious changes in blood vessel network, hypoxia level, or deposition approaches to provide insights into the mechanisms by which the BM of extracellular matrix components, except for collagen IV as expected niche microenvironment controls myelopoiesis. for the fibrotic BA mouse BM niche12 (Extended Data Fig. 2c). We also localized mature populations in relation to cGMPs, and although some Imaging GMPs in situ lymphoid cells were detected inside and at the periphery of cGMPs, − We took advantage of the phenotypic characteristics of GMPs (Lin myeloid cells were only found tightly surrounding cGMPs (Extended + − + + 5 c-Kit Sca-1 FcγR CD34 ) to design an immunofluorescence staining Data Fig. 2d). These results indicate that leukaemic myelopoiesis is scheme allowing their specific recognition on isolated populations and characterized by the presence of cGMPs, which are part of a continuum bone sections (Extended Data Fig. 1a). In wild-type mice, GMPs were of differentiation separating progenitors from mature myeloid cells. 1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Medicine, Division of Hematology/Oncology, University of California San Francisco, San Francisco, California 94143, USA. 2Cambridge University Department of Haematology, Cambridge Institute for Medical Research, Wellcome Trust and MRC Cambridge Stem Cell Institute, Hills Road, Cambridge CB2 0XY, UK. 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, and Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. †Present address: Columbia Stem Cell Initiative, Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032, USA. *These authors contributed equally to this work. 6 APRIL 2017 | VOL 544 | NATURE | 53 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. RESEARCH ARTICLE WT BM BA BM a b c Ctrl BM BA BM a b 5-FU + 1 h GMP BrdU/EdU (1 h BrdU) R R D8–D14 ° ° Ctrl post-5-FU Fc Fc WT mice 37 ± 3 50 ˜m 50 ˜m 80 80 1 h EdU c-Kit c-Kit LSK (5-FU) GMP (5-FU) *** 60 *** 60 BA 40 40 * ** 39 ± 1 4 pH3 Lin/Sca-1/CD150 Lin/Sca-1/CD150 1 h BrdU (%) 1 h BrdU 4 * 400 ˜m 10 ˜m 400 ˜m 10 ˜m 20 5 20 Bone Bone 50 ˜m 5-FU 6 5 6 6 4 4 6 c 4 PB 80 BM (×106) d 8 LSK (×105) 16 GMP (×105) 40 Gr (×106) 0 (D): 0 8 10 12 14 0 8 10 12 14 BrdU c-Kit Lin/Sca-1/CD150 ) *** –1 3 60 6 12 30 GMP l d BA BM e ˜ (5-FU) 100 c-Kit Fc˛R M Gr 2 40 4 8 20 * **6 80 * ** 6 8 Cell number 7 Cell number 10 Myeloid (K 20 10 ** 1 *** 2 4 ***6 3 60 *** ° 6 ° ° 5-FU 8 5 10 5 5 10 10 10 8 7 7 6 0 5-FU 0 0 Merge 40 0 8 10 12 14 (D): 0 8 10 12 14 0 8 10 12 14 0 8 10 12 14 (D): 0 102030 Gr Colony (%) e 5-FU BM M 20 ••• GM •• D0 D8 D8.5 D9 1 ˜m 5-FU Lin/Sca-1/CD150 GM (D): 0 8 10 12 14 R 5-FU ° –1 (150 mg kg ) Fc Figure 2 | GMP clusters are foci of differentiation. a, Proliferation in * c-Kit 5-FU-treated wild-type mice showing experimental scheme and BrdU/EdU incorporation in LSK cells and GMPs (n = 3). b, BrdU incorporation WT mice D9.5 D10 D12 D14 in GMPs from control (Ctrl) and BA mice (n = 3). c, Representative immunofluorescence staining showing myeloid progenitors (red) in D8–D14 + + post-5-FU relation to proliferating EdU (green, top) and dividing pH3 (green, Lin/Sca-1/CD150 10 ˜m bottom) cells in BM from control and BA mice. d, Representative staining of GMP cluster periphery (dotted lines), highlighting differentiating GMPs (arrows) as they lose FcγR expression and gain lineage marker expression. Figure 1 | GMP clusters in leukaemic and regenerative myelopoiesis. e, Single-cell clonogenic methylcellulose analyses of GMPs isolated from a, b, Representative immunofluorescence staining showing GMPs (purple) 5-FU-treated wild-type mice (n = 4). Results are percentage of colony in the BM of wild-type (WT) (a) and diseased Scl-tTA::TRE-BCR/ABL types (asterisks denote significance versus day 0 plating efficiency; filled (BA) (b) mice. Solid lines indicate bone surface, dotted lines denote circles versus granulocyte/macrophage colonies; open circles versus cGMPs, and arrowheads indicate individual GMPs. c, Peripheral blood macrophage colonies), and pictures show representative colonies (PB) (n = 5) and BM regeneration in 5-FU-treated wild-type mice. (GM, granulocyte/macrophages; M, macrophages). Dotted lines denote D, day. d, Changes in haematopoietic populations at the indicated cGMPs. Data are mean ± s.d. (grey bars, reference range). * P ≤ 0.05, days after 5-FU treatment. Gr, granulocytes. e, Representative ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t-test). immunofluorescence staining showing GMPs (purple) in the BM of 5-FU-treated wild-type mice. Stars indicate pGMPs and dotted lines denote cGMPs. Data are mean ± s.d. (grey bars, reference range). individual HSCs undergoing regenerative or leukaemic myelopoiesis ** P < 0.01, *** P < 0.001 (Student’s t-test). (Supplementary Information, Extended Data Fig. 5a–d). Collectively, these results indicate that transient cGMP formation is a hallmark cGMPs are features of myeloid expansion characteristic of emergency myelopoiesis. To determine whether cGMPs could be detected during emergency myelopoiesis, we used several models of myeloid regeneration or cGMPs are foci of myeloid differentiation active myeloid cell production.