In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium
Laurent Yvernogeau ORCID: orcid.org/0000-0002-1752-62151 nAff7,
Rodolphe Gautier1 na1,
Laurence Petit1 na1,
Hanane Khoury1,
Frédéric Relaix ORCID: orcid.org/0000-0003-1270-14722,
Vanessa Ribes3,
Helen Sang4,
Pierre Charbord ORCID: orcid.org/0000-0002-2254-33001,
Michèle Souyri1 nAff8,
Catherine Robin ORCID: orcid.org/0000-0002-9895-90465,6 &
Thierry Jaffredo ORCID: orcid.org/0000-0002-8724-32611
Nature Cell Biology volume 21, pages 1334–1345 (2019)Cite this article
Haematopoiesis
Haematopoietic stem cells
It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.
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Fig. 1: Somite-derived ECs do not contribute to aortic haematopoiesis but are instrumental in the vascularization of the limbs and bones.
Fig. 2: The chicken BM vascular network derives from the somite and produces a cohort of HSPCs during late embryonic life.
Fig. 3: Emergence of CD41+ haematopoietic progenitors from GFP+ ECs in the BM.
Fig. 4: HSPCs derived from the late fetal BM endothelium persist after hatching.
Fig. 5: Conditional expression of YFP in VE-cadherin cells in BM at birth discloses a contribution of vascular ECs to MPPs and Lin−Sca1+c-kit+CD150+CD48− HSCs.
Fig. 6: BM vascular EC-derived HSPCs display an endo-haematopoietic transcriptomic profile and show long-term multilineage reconstitution.
Microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE122857, GSE133804 and GSE133812. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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We apologize to those investigators whose important work we were unable to cite or describe due to space constraints. We thank D. Traver for critical reading of the manuscript and S. Gournet for help in image preparation. We are indebted to R. Adams for his sharing of the Tg(Cdh5Cre/ERT2) transgenic mouse line and to S. Germain for providing us with the founders. We thank the Cell Imaging and Flow Cytometry facility of the IBPS (Paris, France) for access and technical support in confocal image acquisition and the Genom'IC platform at Cochin Institute, Paris for their invaluable help with transcriptomic samples treatment. This work was supported by CNRS, UPMC, Fondation Les Treilles (L.Y.), an EMBO short-term fellowship (L.Y.), MERI and FRM PhD fellowships (H.K.) and grants from FRM (DEQ20100318258) and an ANR/CIRM joint grant (ANR/CIRM 0001–02) in T.J.'s laboratory. The production of the GFP+ chicken embryos was supported by grants from BBSRC and the Wellcome Trust. Part of the work and L.Y. were supported by a European Research Council grant (ERC, project no. 220-H75001EU/HSCOrigin-309361) and a TOP subsidy from NWO/ZonMw (912.15.017) in C.R.'s laboratory.
Laurent Yvernogeau
Present address: Hubrecht Institute, Utrecht, Netherlands
Michèle Souyri
Present address: UMRS 1131, Institut Universitaire d'Hématologie, Hôpital Saint Louis, Paris, France
These authors contributed equally: Rodolphe Gautier, Laurence Petit.
IBPS, Laboratoire de Biologie du Développement CNRS UMR7622, Inserm U1156, Sorbonne Université, Paris, France
Laurent Yvernogeau, Rodolphe Gautier, Laurence Petit, Hanane Khoury, Pierre Charbord, Michèle Souyri & Thierry Jaffredo
Institut Mondor de Recherches Biomédicales, Créteil, France
Frédéric Relaix
Institut Jacques Monod, Paris, France
Vanessa Ribes
The Roslin Institute and R(D)SVS, University of Edinburgh, Edinburgh, UK
Helen Sang
Hubrecht Institute, Utrecht, Netherlands
Catherine Robin
Regenerative Medicine Center, University Medical Center Utrecht, Utrecht, Netherlands
Rodolphe Gautier
Laurence Petit
Hanane Khoury
Pierre Charbord
Thierry Jaffredo
L.Y. and T.J. conceived the project. L.Y. and T.J. designed the experiments on chicken embryos, performed chicken somite grafts and interpreted data. L.Y. performed chicken grafts, carried out immunohistological analysis and performed FACS analysis. R.G. performed chicken graft analysis. L.P. performed all mouse work, including transplantation studies, and analysed the data. H.S. provided GFP transgenic chickens. V.R. and F.R. provided the Pax3CRE X Rosa mTmG mice and critical associated materials. H.K. analysed the YFP+ transcriptome versus the YFP− transcriptome. P.C. analysed transcriptomes, performed network analyses and interpreted data. M.S. designed Pax3CRE X ROSAmTmG and the initial VECAD-Cre ERT X Rosa 26 approaches, analysed the data and provided the additional mouse embryonic and BM adult transcriptomes. C.R. and L.Y. designed and supervised the experiments on adult chickens and the live imaging on sections. T.J. wrote the manuscript. L.Y. and C.R. provided significant input for data analyses, findings, interpretations and manuscript writing.
Correspondence to Thierry Jaffredo.
Extended Data Fig. 1 PSM removal experiment.
a, E2 (15-somite stage, HH11/12) wild-type chicken embryo submitted to a PSM ablation. White arrows located the regions of transverse histological analysis at two representative anterior and posterior levels displayed in 1 and 2. MEP21 (red) and DAPI (blue) staining showed that only the PSM was removed without disturbing the surrounding tissues (10 chicken embryos). b, Whole mounts of two PSMs isolated from six E2 (15-somite stage, HH11/12) wild-type embryos and stained with DAPI (blue), MEP21 (red) and CD45 (green) antibodies, showing that PSMs do not contain any ECs or HCs after dissection. Two independent examples are shown (#1, #2) (6 chicken embryos). c, Mid trunk, thick transverse section of an E3 chicken embryo stained with MEP21 (red) and CD45 (green) antibodies shown as positive control of the immunostainings. Of note, PSMs (b) and thick slice (c) were stained at the same time, using the same antibody solutions. d, Representative FACS analysis on single PSMs isolated from 8 independent 13-15 somite stage embryos showing no contamination by haematopoietic (CD45) or endothelial (MEP21) cells. Controls are two whole embryos without PSMs showing the presence of CD45+ haematopoietic and MEP21+ endothelial cells. Ao, Aorta; Ec, Ectoderm; En, Endoderm; Nc, Notochord; NT, Neural Tube; PSM, Presomitic mesoderm; So, Somatopleura; Sp, Splanchnopleura. Scale bars, 200µm in a, 25μm in a1, a2; 50μm in b and c.
Extended Data Fig. 2 GFP+CD45+ haematopoietic cells in circulating blood of the grafted or control embryos with age.
a-f, CD45 expression in the sample (left) and GFP plot in the CD45+ population (right). a-d, chimeric embryos. a, E4 embryo. b, E8 embryo. c, E12 embryo. d, E16 embryo. Of note, only E16 embryos show GFP+ cells within the CD45+ fraction. e, E12 GFP transgenic embryo. f, E12 WT embryo. 23 chicken embryos analysed. See Extended Data Table 1 for details. Data representative of 4 independent experiments.
Extended Data Fig. 3 GFP+ somite-derived cells contribute to E16/E17 bone marrow CD45+ haematopoietic production.
a-c, Representative fluorescent cross section of the limb bone showing the co-distribution of GFP+ sinusoid cells and CD45+ cells. b, Enlargement of the bone marrow showing GFP+CD45+ cells lining the sinusoids. c, Enlargement of the frame in b, showing the double positive cells. d-l, Series of confocal cross sections separated by 1µm, showing a GFP+CD45+ cell (white arrow). d, g, j, CD45 staining. e, h, k, GFP signal. f, i, l, Merged CD45, GFP and DAPI. Right and bottom banners correspond to YZ and XZ projections of the confocal image, respectively. Experiments were repeated 16 times with similar results (see Extended Data Table 1 for details). Confocal analysis was performed 5 times with similar results. Scale bars in a, 200µm; in b, 40µm; in c, 15µm; in d-l, 20µm.
Extended Data Fig. 4 Flow cytometry analysis reveals the presence of endothelial and haematopoietic cells expressing GFP in the E16-grafted chicken BM.
Distribution of CD45, MEP21, CD41, c-KIT, and CD3 populations in grafted (a, b) or wild-type (c, d) BM mononucleated cells. a, n = 12 independently grafted animals were used for CD45, CD41, c-KIT and CD3 expression and n = 8 for MEP21. b, Representative flow cytometry analysis of an E16/E17 grafted BM showing that GFP+ cells derived from the PSM graft contributed to all lineages. Numbers in green indicate the percentage of GFP+ cells in each population (that is in CD45+, MEP21+, CD41+, c-KIT+ and CD3+ populations). c, n = 3 independent WT animals were used for CD45, CD41, c-KIT expression analysis, n = 2 animals were used for MEP21 and CD3 expression analysis. d, Representative flow cytometry analysis of an E16/E17 WT BM. Data in b representative of 4 independent experiments, 3 in d. Error bars are mean ± SEM.
Extended Data Fig. 5 PSM-derived cells provide haematopoietic precursors able to colonize the secondary haematopoietic organs of E16/E17 grafted chickens.
a, f, i, Global views of the thymus (a), spleen (f) and bursa of Fabricius (i) of an E16 chicken colonized by GFP+ PSM-derived grafted cells. a, Most of the thymus lobes were colonized by GFP+ cells (3 chickens). b, Immunostaining (with DAPI) of a thymus transverse section. c-d, Higher magnification of the frame in (b). GFP (c) and merge of DAPI and GFP (d). e, Flow cytometry analysis of an E16/E17 thymus (2 chickens) revealed the contribution of GFP+ PSM-derived cells to the whole thymus and more precisely to the T lineage (GFP+CD4+). g, Higher magnification of the frame in (f), showing dispersed GFP+ cells in the spleen (2 chickens). h, Flow cytometry analysis of the spleen, showing the contribution of GFP+ PSM-derived cells to the whole spleen and more precisely to the CD45+ haematopoietic lineage (2 chickens). j, Higher magnification of the frame in (i) showing dispersed GFP+ cells in the bursa (2 chickens). k, Flow cytometry analysis showing that the bursa of Fabricius, located far away from the PSM graft, was also colonized by PSM-derived haematopoietic progenitors that derived from the graft (2 chickens). Drawings are shown on the left side of the organ pictures to locate the thymus, spleen and bursa of Fabricius in an E16/E17 chicken. Scale bars in a, b, f, i: 100µm.
Extended Data Fig. 6 Flow cytometry analysis reveals the presence of endothelial and haematopoietic cells expressing GFP in adult BM grafted chickens.
Distribution of CD45, MEP21, CD41, c-KIT, CD3 and KUL1 populations in grafted (a, b) and wild-type (c, d) adult BM mononucleated cells. a, Grafted chickens. CD45 (n = 7), MEP21 (n = 4), CD41 (n = 7), c-KIT (n = 7), CD3 (n = 7) and KUL1 (n = 6) independent chickens were used for the analysis. b, Representative multilineage flow cytometry analysis of a grafted adult BM showing that GFP+ cells derived from the PSM graft contributed to all lineages. Numbers in green indicate the percentages of GFP+ cells in each population (that is in CD45+, MEP21+, CD41+, c-KIT+, CD3+ and KUL1+ populations). c, Wild-type animals. CD45 (n = 5), CD41 (n = 4), c-KIT (n = 5), CD3 (n = 4) and KUL1 (n = 4); independent animals were used for the analysis. n = 2 animals were used for MEP21 expression analysis. d, Representative multilineage flow cytometry analysis of a wild-type adult BM showing the absence of GFP staining. Error bars represent mean ± SEM. Data in b and d representative of 4 independent experiments.
Extended Data Fig. 7 Pax3+ somite-derived cells do not contribute to the aorta haematopoieisis but contribute to the endothelial and haematopoietic lineages in the late fetal BM.
a, Transverse section through the aorta of an E10.5 Pax3 GFP+ mouse embryo at the mid-trunk level. CD31 (red, left), Pax3-GFP (green, middle) double staining counterstained with DAPI (blue). Right panel represents the merge (5 mice). b, Cross section of a Pax3KICRE; ROSA GFP embryo at E8.5 showing that this model tags the somite and recapitulates the Pax3 GFP expression at the same stage (5 mice). c, Flow cytometry analysis of the endothelial and haematopoietic lineages in the BM of Pax3-Cre/mTmG fetuses at E19-E21. n = 3 fetuses from 3 independent experiments. d, Flow cytometry analysis showing GFP+ cell (that is somite-derived cell) contribution to the different endothelial and haematopoietic populations. Mouse somite-derived cells mostly contributed to fetal/new-born BM CD3+, Sca1+c-Kit+ and CD144+CD45+ cell populations. n = 3 foetuses from 3 independent experiments. e, Representative GFP expression in mononucleated BM cells. f, Representative flow cytometry analysis of the mononucleated BM cells revealed GFP+ cells mainly in the CD144+CD45+ population. g, Flow cytometry analysis showing the presence of GFP+ cells in the Sca1+c-KIT+ population (that is HSPC population). h, Negative controls are non-recombined fetuses. Data represent mean ± SEM. Data in e-h representative of 2 independent experiments. Scale bars 350µm in a; 200µm in b.
Extended Data Fig. 8 YFP expression on negative controls and YFP tracing following induction in VE-cadherin+ cells at different time points after birth.
a, Flow cytometry analysis for the presence of YFP+ cells in the CD45+ fraction of the BM immediately after the last tamoxifen injection. Representative new-born mice injected with tamoxifen at post-natal days 1, 2, and 3 and analysed for the expression of CD45 and YFP at day 4. No CD45+ YFP+ cells were found indicating the absence of contamination by haematopoietic progenitors expressing VE-Cadherin (11 animals in 3 independent experiments). The gating strategy is shown for mouse #1. b, c, BM from a representative non-induced mouse at 27 post-natal days. b, Representative flow cytometry analysis showing the absence of YFP expression in mononucleated BM cells and in the CD144−CD45+, CD144+CD45+ and CD144+CD45− cell fractions (4 animals in 2 independent experiments). c, Representative flow cytometry analysis of the mononucleated BM cells showing the absence of YFP expression in the Sca1+c-KIT+ population (that is HSPC population) and in the different HSC, HPC and MPP populations (2 independent experiments). d, Scheme showing the activation of YFP in VE-Cadherin+ (CDH5) cells by tamoxifen injection at different time points after birth (coloured arrows). e, Analysis of the percentages of total YFP+ cells, and YFP+ cells in CD144+CD45− and LSK populations at 21 days post tamoxifen injection. Of note, the x axis values represent the age of the mice from the time of tamoxifen injection. n = 3 mice for d1, n = 6 for d10, n = 7 for d21, n = 7 for d36, n = 5 for d68. Error bars are mean ± SEM. Data in a-c representative of 2 independent experiments.
Extended Data Fig. 9 Haemogenic potential of BM ECs and transcriptome characterisation of the YFP+ and YFP− LSK cells.
a, representative flow cytometry profile showing the gates used to isolate the CD144+CD45− and the CD144−CD45+ populations. Image representative of 7 mice. b-e, representative pictures of the CD144−CD45+ (b) and CD144+CD45− (c, d) cells from 8-day old mouse bone marrow in culture in the endothelial/haematopoietic medium. A high number of floating cells are present in the CD144−CD45+ cells fraction (b). Flat adherent cells (in c) and round, floating, haematopoietic-like cells (arrows in c and d) are present in the CD144+CD45− cell fraction after 4 days of culture. Image representative of 3 experiments. Bar = 10µm. e, f, representative FACS analysis of the CD45+ populations generated after 4 days of culture from the CD144−CD45+ (e) and the CD144+CD45− (f) cell populations. Image representative of 3 experiments. g, PCA with the entire set of mRNAs (30,922 genes) as variables and the basic set of YFP+ (green) and YFP− (red) LSK cells as observations. The two types of transcriptomes were strongly separated (3 biological replicates per population). h, Major GO categories given by DAVID for the gene sets up-regulated in YFP+ (green) and YFP− (red) LSK cells. i, Hierarchical clustering obtained with the 23 samples (3 biological replicates per population except for HC BM and LSK CD150+ BM where quadruplicates were used) as observations and the gene set of 2056 DEGs (986 up, 1070 down) as variables was generated from the PCA displayed in Fig. 6a, bottom panel. Branch organisation reflects the association between the different samples displayed on the PCA. Scale Bars: 50µm in b, c, d.
Extended Data Fig. 10 Long-term repopulation analysis in the peripheral blood of recipients transplanted either with YFP− or YFP+ LSK cells.
a, b, Flow cytometry analysis of circulating blood from 6 (a) and 4 (b) C57Bl/6-CD45.1 recipient mice at 11 weeks injected with 2,000 YFP− (a) or YFP+ (b) LSK cells (C57Bl/6-CD45.2), respectively. Mice recipients transplanted with YFP− LSK cells displayed a more robust reconstitution than the ones transplanted with YFP+ LSK cells. Of note, two mice in the LSK YFP+ series died before two weeks post-injection. c, Back-gating within the GFP+ population of the mononucleated bone marrow cells from mouse #d at 14 weeks of reconstitution. Dot plot representation of flow cytometry multilineage analysis showing Gr1 (granulocytes), CD11b (macrophages/monocytes), B220 and CD19 (B cells), CD4 (T cells), CD8 (T cells), Ter119 and CD71 (red cells), CD41 and CD61 (megakaryocytes) staining. The YFP population was more prominent in B220, CD19, CD71, Gr1 and CD8 fractions. Data in a-c representative of 2 independent experiments.
Supplementary Video 1
Whole-mount staining of E16 chicken bone marrow after GFP+ PSM graft. The bone marrow was stained with anti-MEP21 (endothelial marker, red), anti-CD45 (haematopoietic marker, blue) and anti-GFP (green, which revealed grafted PSM-derived cells) antibodies. The marrow was heavily colonized by GFP+ PSM-derived cells that contributed to the whole bone marrow vascularization (n = 3).
A GFP+ endothelial cell co-expressing CD45 as revealed by confocal analysis of an E16-grafted BM. Series of confocal sections separated by 1 µm, showing a GFP+CD45+ cell (white arrow). Panels: upper left, DAPI; upper right, CD45; lower left, GFP; lower right, merged. Right and bottom banners correspond to y–z and x–z projections of the confocal image, respectively (n = 5 in three independent embryos).
GFP+ endothelial cells co-expressing CD41 are integrated in the vascular endothelium of E16-grafted BM. Series of confocal sections of 1 µm, showing a GFP+CD41+ cell integrated in the GFP+ vascular endothelium (n = 8 in three independent embryos).
Emergence of CD41+ haematopoietic precursors from GFP+ endothelial cells in the bone marrow. a, Time-lapse live imaging of a transversal E16 bone marrow slice showing the emergence of a GFP+ cell from the endothelium of a blood vessel. b, After time-lapse imaging (a) the section was stained again with anti-CD41-PE antibodies. The newly emerged GFP+ cell expressed the haematopoietic marker CD41. Of note, this cell was CD41- at the beginning of imaging. Time is in hours and minutes (n = 2 independent experimental animals with five different sections per slide). Scale bar, 15 µm.
Whole-mount staining of an E16 chicken thymus after GFP+ PSM graft. The thymus was stained with anti-MEP21 (endothelial marker, red), anti-CD45 (haematopoietic marker, blue) and anti-GFP (green, which revealed grafted PSM-derived cells) antibodies. The thymus was colonized by GFP+ PSM-derived cells, some of the lobes being more colonized than others. GFP+ cells could colonize secondary haematopoietic organs that are located far from the grafting site. (n = 2 independent experimental animals).
Extended Data Table 1
Number of grafted chickens, time points of analyses and type of analyses performed. This table summarizes the number of chicken samples according to the age at analysis; the organs or tissues analysed, that is, the thymus, bone marrow, spleen; the kind of analysis applied, that is, FACS, immunostaining, confocal imaging or videos; and the number of cases analysed for each time point.
List of anti-mouse and anti-chicken antibodies. List of anti-mouse (first) and anti-chicken (second) antibodies used in the study, including the conjugated fluorochrome when applicable, clone of origin, cell specificity, supplier and catalogue number.
Source Data Fig. 2
Statistical source data
Source Data Extended Data Fig. 4
Yvernogeau, L., Gautier, R., Petit, L. et al. In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium. Nat Cell Biol 21, 1334–1345 (2019). https://doi.org/10.1038/s41556-019-0410-6
Issue Date: November 2019
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