Progressive maturation toward hematopoietic stem cells in the mouse embryo aorta

All blood cell production throughout adult life originates from hematopoietic stem cells (HSCs) that are initially produced during embryonic development. HSCs are first detected in the aorta of the aorta-gonad-mesonephros (AGM) region at embryonic day(E)10.5 of mouse development.¹  They most likely reside in cell clusters—intra-aortic hematopoietic clusters (IAHCs)—tightly attached to the endothelium. Indeed, both HSCs and IAHC cells express similar markers (eg, c-kit² ) and are absent in Runx1^−/− embryos.³  IAHCs, first described a century ago,⁴  are present in most vertebrate species and believed to derive from the underlying endothelium.⁴  The so-called “hemogenic endothelial origin” of IAHCs and HSCs was shown in chicken⁵  and mouse embryos.^6-9  It was definitively confirmed after we and others observed the endothelial to hematopoietic transition directly in the aorta by time-lapse confocal microscopy, ex vivo in thick mouse embryo slices,¹⁰  and in vivo in zebrafish embryos.^11,12 

Some discrepancies remain when HSCs and IAHCs are compared: (1) IAHCs appear 1 day before HSCs (E9.5 and E10.5, respectively)^1,2 ; (2) the number of IAHC cells largely exceeds the number of HSCs estimated per AGM (∼600 IAHC cells and <0.1 HSC at E10.5; ∼500 IAHC cells and <2 HSCs at E11.5^1,2,13 ); and (3) although IAHCs are located in both sides of the aorta, HSCs are restricted to the ventral side.¹⁴  To determine the exact cell composition and function of IAHCs, we analyzed the phenotypic evolution and function of IAHC cells before and during HSC detection. Here we show that IAHCs contain pre-HSCs (or HSC precursors) capable of long-term multilineage reconstitution in newborn recipients at a time when HSCs are not yet detected. We demonstrate that IAHC pre-HSCs mature toward an HSC fate by performing secondary transplantations in adults and ex vivo time-lapse live confocal imaging.

IAHCs were analyzed by scanning electron microscopy or after immunostaining on embryos (sliced or whole). Sorted IAHC cells were tested in vitro in hematopoietic progenitor assays and in vivo after primary long-term transplantation in Rag2^−/−γc^−/− or wild-type (WT) newborns. Secondary transplantations were performed in adult Rag2^−/−γc^−/− or WT recipients. Donor chimerism was analyzed by flow cytometry 4 to 5 months upon transplantation.

To observe in detail the structure of IAHCs, we imaged by scanning electron microscopy E10 (Figure 1A-C and supplemental Figure 1C-G, available on the Blood Web site) and E11 (supplemental Figure 1A-B) embryo slices. IAHCs, endothelium and subaortic mesenchyme were clearly visible (Figure 1C). In addition to observing single cells (supplemental Figure 1B), we observed spheroid (Figure 1C), stack (supplemental Figure 1C), and “mushroom” (supplemental Figure 1D) IAHC shapes. Most cells were spherical with surface microvilli (supplemental Figure 1E-G). As a control, no IAHCs were visible in Runx1^−/− embryo slices (supplemental Figure 1H-I).

The functional characterization of IAHCs has been hindered by the difficulty to isolate IAHC cells to purity. All IAHC cells express c-kit, but contaminating c-kit⁺ cells are also present in the circulating blood and outside of the aorta.^2,15  To circumvent this issue, directly-labeled antibodies against c-kit were injected inside the aorta of nonfixed E10 embryos before AGM dissection/dissociation. Our procedure allows flushing out the blood from the aorta, staining all IAHCs,^10,16  and isolating IAHC cells only (Figure 1D). When the AGM is dissociated before staining, contaminating c-kit⁺ cells will also be stained (Figure 1E). Therefore, c-kit intraaortic staining allows IAHCs isolation to purity.

c-kit⁺ IAHC cells express several endothelial and hematopoietic surface markers (supplemental Figure 2A-D [E10] and 2E-H [E11]). Ly6a-GFP and CD45 were clearly differentially expressed in IAHCs.^2,10,16,17  It revealed 4 phenotypically distinct populations (c-kit⁺Ly6a-GFP^–CD45^–, c-kit⁺Ly6a-GFP⁺CD45^–, c-kit⁺Ly6a-GFP^–CD45⁺, and c-kit⁺Ly6a-GFP⁺CD45⁺), as shown by flow cytometry or confocal microscopy at E10 (Figure 1F, supplemental Figure 3, and supplemental Movie 1) and E11 (supplemental Figure 4A).

To test whether IAHCs contain committed hematopoietic progenitors and whether these progenitors might be restricted to a particular phenotype, IAHC cells were tested in vitro in 3 different clonogenic assays for the presence of erythroid/myeloid progenitors, pre–B-lymphoid progenitors, or megakaryocyte progenitors (colony-forming unit [CFU-Mk]). We found that IAHCs contained very few erythroid/myeloid progenitors (13 ± 3 erythroid/myeloid progenitors/E10 AGM, n = 3, Figure 1G; 19 ± 4 erythroid/myeloid progenitors/E11 AGM, n = 3, supplemental Figure 4B), very few CFU-Mk (9 ± 2 CFU-Mk/E10 AGM, n = 2), and no pre–B progenitors (neither in E10 c-kit⁺ yolk sac sorted cells, data not shown). As a positive control, adult bone marrow cells produced pre–B progenitors in our culture system (data not shown). Of note, B progenitors were previously reported in the AGM, but only after a culture step on stromal cell lines.¹⁸  The number and types of myeloid progenitors were similar in the 4 sorted IAHC cell populations, indicating that a particular phenotype does not correlate with a specific type of progenitor at E10. Interestingly, most erythroid/myeloid progenitors at E10 and E11 were not present in IAHCs but in the AGM circulating blood (enriched in the c-kit⁺Ly6a-GFP^–CD45⁺ fraction) (supplemental Figure 4C-D and 4E-F, respectively), most likely coming from the yolk sac.¹⁹  Altogether, our data show that HSCs and committed progenitors do not account for the high IAHC cell number.

We then tested whether IAHCs contained pre-HSCs (HSC precursors). Pre-HSCs were previously reported in precultured E8 para-aortic splanchnopleura (P-Sp) (prospective AGM area),²⁰  E9.0 yolk sac, and P-Sp.^21,22  These cells, unable to reconstitute adult, irradiated WT recipients, could reconstitute more permissive recipients (eg, busulfan-conditioned WT neonates, immunodeficient adult). Secondary transplantations performed in WT adult recipients also confirmed the self-renewal potential of the pre-HSCs.²¹  Pre-HSCs were also identified after an in vitro AGM-dissociated/reaggregated culture.^23,24  We performed transplantations of c-kit⁺ IAHC cells isolated from early E10 (before HSC detection) and mid-E10 (beginning of HSC detection) AGMs into the liver of Rag2^−/−γc^−/− or WT neonates (Table 1). Early E10 IAHC cells reconstituted at long-term both Rag2^−/−γc^−/− and WT neonate recipients after the minimum injection of 100 and 200 cells, respectively. Mid-E10 IAHC cells reconstituted both types of recipients with as few as 50 cells. All reconstitutions were multilineage with donor contribution in myeloid, T-, and B-lymphoid lineages (Figure 1H and supplemental Figure 5). Thus, IAHCs contain pre-HSCs, and their frequency increases between early and mid-E10. Based on the number of c-kit⁺ cells (∼200 and 600 c-kit⁺ cells/aorta at early E10 and mid-E10, respectively)²  and our neonate transplantation data (all recipients reconstituted when injected with 100 and 50 c-kit⁺ cells from early E10 and mid-E10 embryos, respectively), we estimate that there is an average of 2 pre-HSCs per aorta at early E10. This number increases to 12 pre-HSCs at mid-E10, which largely exceeds the number of HSCs estimated at this stage (∼0.1 HSC).^1,13  All 4 c-kit⁺ IAHC subpopulations reconstituted Rag2^−/−γc^−/− neonates (Table 1). Therefore, pre-HSCs in IAHCs are not restricted to a specific phenotype at E10, based on Ly6a-GFP and CD45 expression. Both markers are thus not useful to enrich in pre-HSCs at E10 but are indicative of pre-HSC maturation into an HSC fate (because all HSCs are Ly6a-GFP⁺CD45⁺ at E11.5).

Our data show that pre-HSCs not only derive from Ly6a-GFP⁺ hemogenic endothelial cells²⁵  but also from Ly6a-GFP^– hemogenic endothelial cells (not visible by fluorescent live confocal imaging¹⁰ ). Ly6a-GFP is thus very unlikely to be an exclusive marker of hemogenic endothelial cells. To date, only Runx1 seems to mark endothelial cells endowed with the potential of producing blood cells.⁶  To determine whether IAHC pre-HSC maturation occurs in vivo, secondary transplantations were performed in adult recipients (Rag2^−/−γc^−/− and/or WT), with bone marrow and spleen cells isolated from primary reconstituted recipients (identified by the paragraph symbol [¶] in Table 1) (supplemental Table 1). In both cases, long-term multilineage reconstitution was observed in the bone marrow, spleen, and peripheral blood of the recipients. Therefore, IAHC pre-HSCs can mature into fully potent HSCs in vivo. Time-lapse confocal microscopy of E10 Ly6a-GFP embryo slices (stained with CD31 and c-kit antibodies before imaging) revealed that, although most IAHC cells were Ly6a-GFP^–, they all began to express GFP during the course of imaging (Figure 1I and supplemental Movie 2), indicative of maturation toward an HSC fate (all HSCs are Ly6a-GFP⁺).¹⁷  To test whether cell proliferation occurred in IAHCs, embryo slices were stained for phospho-histone H3.3 (PHH3), CD31, and c-kit (supplemental Figure 6A-B), and proliferating IAHC cells (c-kit⁺PHH3⁺) were counted to determine the mitotic index (percentage of IAHC proliferating cells per total IAHC cells; Figure 1J). Few IAHC cells proliferated at the early, mid, and late E10 stages. Thus, the majority of IAHC cells mature toward a putative HSC state with low proliferation.

The hemogenic endothelium does not directly form fully potent HSCs but an intermediate cell population organized in IAHCs. Concomitant to HSC detection (mid-E10), IAHCs already contain an average of 12 pre-HSCs and 22 committed progenitors. The vast majority of IAHC cells is therefore not yet identified but could be very immature pre-HSCs not yet able to reconstitute neonate recipients. We hypothesize that IAHC cells will progressively become pre-HSCs, maturing toward an HSC fate via successive steps initiated inside the aorta and possibly completed in the fetal liver (main HSC reservoir at midgestation) (supplemental Figure 8). We indeed observed 3 phenotypically defined IAHC pre-HSC populations in E12 fetal liver (supplemental Figure 7A-B). To which extent the pre-HSC populations described thus far in different hematopoietic sites are related remains to be determined.

The authors thank the experimental animal center (Experimental Dieren Centrum, Erasmus University Medical Center) for mouse care and the Optical Imaging Center of the Erasmus MC for confocal microscope access, Reinier van der Linden and Julien Karrich for help with cell sorting, Elize Haasdijk for initial help with the vibratome, Niels Galjart (Erasmus University Medical Center) for access to the Volocity software, Fanny Sage for her help in editing the movies, and Jacqueline Deschamps (Hubrecht Institute) for careful reading of the manuscript.

This work was supported in part by a European Research Council grant (project number 220-H75001 EU/HSCOrigin-309361); an Innovational Research Incentives Scheme (NWO-VIDI) grant (917-76-345) (C.R., J.-C.B.), and Landsteiner Foundation for Blood Transfusion Research (LSBR 1025) (T. Clapes).

Contribution: C.R. and J.-C.B. conceived ideas and designed the research; J.-C.B. performed most experiments with the help of C.R., T. Clapes, and A.K.; J.O. performed scanning electron microscopy, supervised by M.M.; N.P. performed the initial neonate transplantations, supervised by T. Cupedo; J.-C.B. and C.R. analyzed the data, interpreted the experiments, created the figures, and wrote the paper; and all authors discussed the results and commented on the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Catherine Robin, Hubrecht Institute–KNAW & and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands; e-mail: c.robin@hubrecht.eu.