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Bone marrow hematons: An access point to the human hematopoietic niche

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R E S E A R C H A R T I C L E

Bone marrow hematons: An access point to the human

hematopoietic niche

Alexandre Janel

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Juliette Berger

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Celine Bourgne

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Richard Lemal

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Nathalie Boiret-Dupre

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Frederique Dubois-Galopin

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Pierre Dechelotte

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Charlotte Bothorel

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Eric Hermet

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Sara Chabi

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Jacques-Olivier Bay

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Celine Lambert

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Bruno Pereira

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Françoise Pflumio

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Rima Haddad

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Marc G. Berger

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1CHU Clermont-Ferrand, H^opital Estaing, Hematologie Biologique, 1 place Lucie et Raymond Aubrac, Clermont-Ferrand Cedex 1, 63003, France;2Universite

Clermont Auvergne, Equipe d’accueil l’EA 7453 CHELTER, 1 place L. et R. Aubrac, Clermont-Ferrand Cedex, 63003, France;3CHU Clermont-Ferrand, H^opital Estaing,

CRB-Auvergne, 1 place Lucie et Raymond Aubrac, Clermont-Ferrand Cedex 1, 63003, France;4CHU Clermont-Ferrand, H^opital Estaing, Hematologie Clinique, 1 place

Lucie et Raymond Aubrac, Clermont-Ferrand Cedex 1, 63003, France;5CHU de Toulouse, H^opital Purpan, Laboratoire d’Hematologie, Place du Docteur Baylac - TSA 40031 31059, Toulouse Cedex 9, France;6CHU Clermont-Ferrand, H^opital Estaing, Anatomie Pathologique, 1 place Lucie et Raymond Aubrac, Clermont-Ferrand Cedex 1, 63003, France;7INSERM UMR967, CEA/DSV/iRCM, Laboratory of Hematopoietic Stem cells and Leukemic Cells, Equipe labellisee par la Ligue Nationale

Contre le Cancer, Universite Paris Diderot, Universite Paris-Saclay, Univ Paris Sud, Commissariat a l’Energie Atomique et aux Energies Alternatives, Fontenay-aux-Roses, 92265, France;8CHU Clermont-Ferrand, Departement de Recherche Clinique et Innovation, Bd Leon Malfreyt, Clermont-Ferrand, France

Correspondence

Marc G. Berger, Hematologie Biologique, CHU Estaing, 1 place Lucie et Raymond Aubrac, 63003 Clermont-Ferrand Cedex 1, France.

Email: mberger@chu-clermontferrand.fr Funding information

“CHU de Clermont-Ferrand”, the EA 7283 CREaT (Cancer Resistance Exploring and Targeting), the GECOMH association and honorarium received by MGB for hematol-ogy expertise. Confocal microscopy training of AJ was supported by a grant from Can-cerop^ole CLARA (Mobilite Des Chercheurs 2010-2011 AXE VI Echappement Tumoral). FP and RH are supported by INSERM, CEA, Universite Paris 7, Universite Paris Saclay, Universite Paris Sud, Ligue Nationale contre le Cancer, Institut du Cancer and Region Ile de France. SC has a salary from Agence Nationale pour la Recherche.

Abstract

To understand the complex interactions between hematopoietic stem cells and the bone marrow niche, a human experimental model is needed. Our hypothesis is that hematons are an appropriate ex vivo model of human bone marrow. We analyzed the hierarchical hematopoietic cell content and the tissue organization of single hematons from healthy donors. Most (>90%) hematons con-tained precursors of all cell lineages, myeloid progenitors, and LTC-ICs without preferential commitment. Approximately, half of the hematons could generate significant levels of lympho-myeloid hematopoiesis after transplantation in an NSG mouse model, despite the low absolute numbers of transplanted CD341 cells. Mesenchymal STRO-11 and/or CD2711 cells formed a critical network that preserved hematon cohesion, and STRO-11cells colocalized with most hema-topoietic CD341 cells (68%). We observed an influence of age and gender. These structures represent a particularly attractive model for studying the homeostasis of the bone marrow niche and pathological changes that occur during diseases.

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I N T R O D U C T I O N

Postnatal hematopoietic stem cells (HSCs) are mainly located in the bone marrow (BM), where their stem cell fate is regulated by a complex network of local interactions thought to be concentrated in the BM niche.1,2HSC niches in BM have been intensively examined because of

their potential involvement in numerous health conditions. A better understanding of the role of these micro-anatomic HSC niche areas may enable the design of new therapeutic strategies. Imaging

techniques such as HSC spatial tracking by multi-photon or confocal microscopy have revealed niche locations, at least in endosteal and vascular areas.3,4In parallel, advancements in molecular tools and cell

identification techniques have enabled the identification of the cells that constitute the niche and potentially play roles as HSC partners in hematopoiesis.5

However, most studies have been conducted in mouse models; although these models are very useful, they do not completely recapit-ulate the human HSC BM niche. Access to the human BM niche

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VC2017 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/ajh Am J Hematol. 2017;92:1020–1031.

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remains problematic. Acquiring BM biopsy samples is relatively simple but reserved for precise indications, and the decalcification process allows for only immunohistological studies. With a BM puncture, mac-roscopic coherent tissue aggregates, termed hematons, are collected in a cell suspension. The qualitative properties of this tissue compartment have been studied,6,7and we previously confirmed its enrichment with long-term culture-initiating cells (LTC-ICs) and primitive CD341cells8;

yet, hematons have not been examined to determine if each cell aggre-gate is a complete ex vivo surroaggre-gate of BM tissue and would constitute a model that can be used to study all aspects of human hematopoiesis. In this study, we evaluated hematons as single tissue structures.

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M E T H O D S

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Human BM and cells

Biological samples were collected from 19 donors (median age: 41 years (range: 17–84 years), 10 male/9 female) (Supporting Information Table S1). First, we saved the typically discarded mesh from healthy donor BM filters (HD). Second, we used the remaining portion of the biological samples collected for diagnosis (DS), which showed normal analysis results (n5 5) (see Supporting Information Table S1). All sam-ples could be used for research because the patients had been informed and did not verbally express any disagreement, as stipulated by French law.

Because of spontaneous dissociation after cryopreservation in DMSO, fresh hematons were isolated<24 h after harvest, and each structure was dissociated as previously described.8Cells were counted,

characterized after cytospinning and May-Gr€unwald-Giemsa staining, and/or used in functional assays.

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Immunohistochemistry

Routine techniques were used for immunohistochemistry, including deparaffinizing, rehydrating, and incubating with antibodies steps. All sections were counterstained before analysis (see Supporting Informa-tion for details).

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Scanning electronic microscopy

Hematons were washed in cacodylate buffer (0.2 M; pH 7.4) for 5 min and then fixed in glutaraldehyde (1.6%) in cacodylate buffer at 48C for 1 h at room temperature. Fixed specimens were washed in cacodylate buffer and postfixed for 1 h at 48C in buffered osmium tetra-oxide, dehydrated, and then metallized. Hematons were examined under a Jeol6060-Low Cacuum electron microscope.

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Confocal microscopy and image analysis

Hematons were fixed in 4% paraformaldehyde overnight at 48C. After 2 washes with PBS/3% BSA, hematons were permeabilized for 2 h in PBS containing 5% human serum, 5% goat serum, 3% BSA, 10 nM HEPES, and 0.1% saponin and then washed with PBS containing 3% BSA, 10 mM HEPES, and 0.1% saponin (called wash medium). Anti

STRO-1 (Clone STRO-1, dilution 1:25, R&D Systems, Minneapolis, MN, USA) antibody staining was performed overnight at 48C. After washing twice with wash medium, the secondary antibody incubation was per-formed for 2 h at 228C (Cy-5 IgM, 1:100 or FITC conjugated anti-IgM, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After two washes with wash medium, phyco-erythrin conjugated CD34 anti-body (AO7776, 1:25, Beckman Coulter, Brea, CA, USA) or anti-CD271 (APC anti-IgG, 1:100; Miltenyi Biotec) was added, followed by incuba-tion overnight at 48C. After sequential washing with wash medium and then PBS/3% BSA and PBS, the hematons were cover-slipped with Hoechst nuclear counterstain (Hoechst 33342; 1/10,000; Invitrogen, Carlsbad, CA, USA).

Confocal images were obtained using a Zeiss LSM510Meta micro-scope with 40X/1.3 Plan-NeoFluar or 63X/1.4 Plan-Apochromat objectives and processed using LSM510Meta Software (Zeiss, Jena, Germany). For 3-D images, a series of 15–30 z-sections taken at a distance of 1mm was acquired for each hematon. All analyses were performed using ImageJ software (NIH, Bethesda, MD, USA).9 Hemato-poietic CD341cells were identified by superimposing each field of the Hoechst (blue) staining with the CD34 (red) after 3-D reconstruction to assess typical hematopoietic cell morphology that could be distin-guished from endothelial cells. Indeed endothelial cells appeared always as elongated cells with acute-angled ends, with small ovoid nuclei as shown with anti-CD31 staining (Data not shown).

To assess the precise anatomical relationships in the niche, we acquired Z-stack images containing the best focal plane for the cell sig-nal and the focal plane of the closest mesenchymal surface, and then, the distances were quantified.10Contact between cells was scored on 3-D reconstructed images by visual inspection. Colocalization between hematopoietic CD341 cells and STRO-11cells was tested using the Poisson distribution.11

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Flow cytometry

The percentage of viable CD341 cells was evaluated using a gating strategy based on the basic ISHAGE protocol (see Supporting Informa-tion for details).

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CFC and CFU-F assays

Cells from each BM hematon (3–8 per BM) were cultured in short-term culture as described previously12(see Supporting Information for

details). In some experiments, mesenchymal cells (MCs) were expanded in vitro through one passage before being assayed in the LTC-IC assay.

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LTC-ICs in a limiting dilution assay (LDA)

We analyzed the hematon content of the LTC-ICs13by seeding all cells

in the MS5 cell line monolayer in LTC media (Stemcell Technologies, Inc., Vancouver, Canada) as previously described.8The only

modifica-tion involved using an LDA method in which different propormodifica-tions of hematon-dissociated cell suspensions were plated (1/8, 1/16, 1/64, and 1/156 in 2, 4, 8, and 16 wells, respectively) because the frequency of CD341cells in each hematon could not be evaluated before LTC.

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Furthermore, to verify that MCs expanded from hematons could sustain hematopoiesis, we tested the capacity of each hematon-derived stroma to sustain LTC-IC proliferation by seeding it with immu-noselected CD341 cells from healthy donors (see Supporting Information).

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Xenotransplantation

NOD.Cg-Prkdc (scid)Il2rg(tm1Wjll)/SzJ (abbreviated NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were housed in pathogen-free animal facilities at CEA (Fontenay-aux-Roses, France). Mice were irradiated with 3 Gy (IBL 637 CisBio International, Codolet, France; dose rate: 0.61 Gy/min) and anesthetized with isoflurane before cells derived from single or pooled/mixed (6–10 per mouse) hematons obtained from individual BM samples were transplanted into their femurs (n5 23 and n 5 9 for single and mixed hematons, respectively) (Supporting Information Table S2) followed by a 3mg/mL buprenor-phine treatment. After 16 weeks, the mice were euthanized by CO2

inhalation using an infusion device (TEM SEGA, Pessac, France). The BM, spleen, and thymus were analyzed by FACS (BDTMLSRII) for the presence of human hematopoietic cells using the conjugated mouse anti–human-specific monoclonal antibodies listed in Supporting Infor-mation Table S3.

Experimental procedures were performed in compliance with the French Agriculture Ministry and local ethics committee regulations (Authorization number 12–015).

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Statistical analysis

Statistical analyses were performed using Stata software (version 13, StataCorp, College Station). The tests were two-sided, with a type I error set ata 5 0.05. Results are expressed as the mean 6 SEM. Random-effects models for correlated measures (data from each bone marrow sample) were used rather than usual statistical tests, which would be not appropriate because the hypothesis regarding the independence of the data was not verified. Intra-class correlations were estimated to measure the within and between bone marrow sample variabilities. When appli-cable, a series of paired experiments were compared using paired Stu-dent’s t-tests. Finally, LDA was performed according to the Poisson statistical model (L-CalcTM, StemCell Technologies).

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R E S U L T S

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A hematon is an organized BM tissue structure

that reflects the BM cell composition

In all cases, hematons appeared as individual coherent cell aggregates that resisted in vitro handling (Supporting Information Figure S1), and they maintained their structure in liquid medium for 2–5 days before disruption. Histological analysis showed that the cells appeared to be held together by multiple membrane extension-producing cells that formed a network (Figure 1A–C; Supporting Information Figure S2); these cells were likely of mesenchymal origin. Hematons exhibited con-nected lobular formations containing hematopoietic cells; occasionally,

a splinter of bone was observed (Figure 1D). In all hematons, we observed adipocytes (Figure 1D–F,J; Supporting Information Video S1) and vascular vessels, and sometimes arterioles were observed (Figure 1F–H,I; Supporting Information Figure S3; Videos S2 and S3).

Investigation of the cellular composition of hematons of 11 BM samples showed that individual hematons contained a limited number of cells (average of 736 10 3 103cells), which varied in intra- and inter-donor manners (range: 4–121 3 103cells; Supporting Information

Figure S4). Then, in 6 single hematons from 4 BM samples, we observed similar cytological profiles of differentiated cells in the differ-ent hematons from the same donor compared with those defined after smear analysis and with all lympho-myeloid precursors in every ana-lyzed structure (Supporting Information Figure S4B).

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Individual hematons contain functional immature

progenitors and primitive cells of the human

hematopoietic cell hierarchy

We next analyzed the CD341cell content of hematons and compared it with that of filtered BM. In all cases, we detected a higher frequency of CD341 cells in hematons (3% vs. 0.96% respectively; Figure 2A; P5 .0045), with a median absolute number of 2.1 3 103CD341cells

per hematon (Figure 2B).

To avoid cell loss, we seeded cells without cell sorting. We detected CFU-GM and BFU-E in 46/53 (86.8%) and 48/53 (90.5%) of single tested hematons, respectively (Figure 2C), with a higher number of CFU-GM. In parallel, 24/28 (85.7%) hematons contained Mk pro-genitors (Figure 2D). Moreover, we also measured the frequency of LTC-ICs. A total of 91% of the 55 tested hematons (n5 6 BM) con-tained LTC-ICs (Supporting Information Figure S5, Figure 2E).

Finally, we transplanted hematon-derived cells into the BM of NSG mice. A total of 23 individual hematons (6–9 mice/experiment) and mix-ture of hematons (6–10 per mouse, 3 mice/experiment) obtained from 3 HDs were tested. Engraftment was considered positive when at least 0.02% of human CD451cells were found to contain B lymphoid and myeloid cells at week 16. These levels were lower than those previously described,14reflecting a relatively low number of CD341 adult cells transplanted into mice. The average percentage and median number of CD341 cells per hematon, extrapolated from a cell suspension from more than 20 hematons, revealed that each mouse was transplanted with less than 23 103CD341cells from each single hematon.

The results showed that 9/21 (42.9%) and 7/8 (87.5%) mice injected with single and mixed hematons, respectively, showed chimer-ism, albeit variable, in the bone marrow, with a median of 0.054% and 0.35% of human CD451cells 4 months after transplantation (Figure 2F, Supporting Information Figure S6A). Importantly, lympho-myeloid cells could be detected in the murine BM, spleen, and thymus (Support-ing Information Figure S6 and Table S1). Engrafted s(Support-ingle or mixed hematons generated both B and myeloid cells in the BM, and in both cases, a higher B cell proportion was detected (Supporting Information Figure S6A, Figure 2F), as is typically observed in NSG mice following transplantation of normal adult or fetal CD341cells.15,16Xenografted mice also harbored splenic lymphoid B and T cells (Supporting

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F I G U R E 1 Hematons are organized structures of bone marrow tissue. Representative examples showing the tissue organization of hematons: (A, details in B, C) Electronic microscopy analysis showing that the structure is supported by a network of cells with numerous cytoplasmic expansions (arrows). This network of cytoplasmic processes is associated with an amorphous structure corresponding to the extracellular matrix (arrowhead). The large spheres are adipocytes (a). Hematopoietic cells (*) are retained within the mesenchymal network (D, details in E, F). Representative sections from hematons after hematoxylin and eosin staining showing that hematons have mostly a lobular structure (L: lobe). We sometimes observed small splinters of bone (D; b: bone). Dense hematopoietic tissue spread out in the available space between adipocytes, corresponding to optically empty rounded regions. All lineages were present, even those that were the most infrequent, such as megakaryocytes (E, arrow head). (F, details in G, H) Another representative section of the hematon shown in D, stained with anti-CD34 antibody, is represented. A small number of cells were positive for CD34 antigen. Some cells correspond to endo-thelial cells (recognizable by their morphology, along vessels, arrow in G), others correspond to immature hematopoietic cells (small, round cells, arrow head in H). (I) Confocal microscopy (red: CD34, blue: Hoechst) confirmed the tissue structure and presence of a vascular net-work in all analyzed hematons, identified by CD34 staining and cell morphology. This picture is the sum of 12 distinct slices 2mm thick. (J) Adipocytes were observed after staining with lipophilic bodipy 493/503 (green) associated with nuclei staining (blue: Hoechst). The confocal microscopy picture is the sum of 15 distinct slices 4mm thick. White bars correspond to 200 (D, F), 100 microns (J) or 50 microns (A, E, G, H, I) or 5 microns (B and C) [Color figure can be viewed at wileyonlinelibrary.com]

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F I G U R E 2 Hematons accommodate all usual functional hematopoietic progenitors and repopulating stem cells representative of hematopoietic cell hierarchy. Hematopoietic progenitor content was assayed at the single-hematon level, from all cells from structure. (A) We used flow cytometry to compare the percentage of CD341in 3–5 hematons from HD BMs (n 5 3) (open square) with the matched fil-tered graft cell suspension (dark circle). All hematons were enriched in CD341cells. Means are indicated as bars. (**: P5 .0045). (B) Next, because of cell content variability, we evaluated the total CD341cell content of hematons, which revealed significantly different contents between donors (*: P< .05). (C, D) In a second series of experiments, we assayed each dissociated hematon in a CFC assay. We detected CFU-GM (dark circle), BFU-E (blank circle) (C) and CFU-Mk (dark lozenge)(D) in most hematons (>85%). (E) Lastly, we seeded cells from each hematon (5–10 per BM, 55 hematons tested) from 6 independent BMs in LTC (see Materials and Methods); we calculated the fre-quencies of LTC-ICs in limiting dilution assay (see Supporting Information Figure S5 for demonstrative example) and their total number in each hematon. We detected LTC-ICs in 91% of hematons (0: no detectable LTC-IC). (F) Graphs representing median of percentages of chi-merism and B lymphoid and myeloid in vivo reconstitution from three independent experiments, circles (䊊), blank squares (w), and black tri-angles (䉱) represent individual mice transplanted with each BM sample. (G) Graphical representation of the main characteristics of the bone marrows used in the transplant experiments in mice. Each axis corresponds to the median content of each bone marrow (one color per source marrow) (BFU-E, CFU-GM and LTIC-IC: median number per hematon; BM, spleen and thymus chimerism: percentage of positive mice from single-hematon transplantation experiment) [Color figure can be viewed at wileyonlinelibrary.com]

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Information Figure S6B). Furthermore, although the presence of T cells in the spleen or BM could have been due to the engraftment of con-taminating mature cells in the injected human cell suspension (observed in HD10), the presence of CD41CD81and CD31 thymo-cytes in the thymus strongly suggests in situ human T cell differentia-tion (HD9 and HD11; Supporting Information Figure S6C). Interestingly, one out of the two mice injected with HD11-derived mixed hematons generated very rare B and myeloid cells in the BM (< 0.4% of CD451 cells, (Figure 2F)), exclusively composed of CD31 T cells related to strong T cell differentiation in the thymus (data not shown). In fact, in vivo thymic T cell differentiation varied between experiments and depended on the injected sample (single vs. mixed hematons). Accordingly, T cell development from a single hematon was only detected in 1/3 of experiments, and as expected, mixed hematons generated T cells more efficiently in 2/3 of experiments (Supporting Information Figure S6C and Table S1). These results indicate that multi-potent T/B/M SRCs are detectable but are rarer in a single hematon. To preserve all single hematon-derived cells for xenograft, we eval-uated the content of hematons using other hematons from the same BM sample (Figure 2G and Supporting Information Table S1).

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Hematons recapitulate a hematopoietic niche for

human immature cells

MCs appear to play a critical role, as we observed that the spontaneous in vitro dissociation of hematons strictly coincided with the adhesion of MCs to the plastic dishes (data not shown). We detected CFU-Fs in 82% of samples (63/77 hematons from 7 BMs; Figure 3). A mean of 78.53 103(range: 0.4

–280 3 103) expanded MCs were collected at

the end of primo-culture, representing an average of 12.1 population doublings, which is similar to our previous results.12 Second, each

hematon-derived stroma (n5 5–10 from each of three distinct BMs) was cocultured with allogenic immunoselected CD341cells under LTC

conditions. We observed that 21/24 stromas efficiently supported human hematopoiesis, with the detection of LTC-ICs among the seeded CD341cells (Figure 3B,C).

Third, using confocal microscopy, we visualized simultaneously STRO-11 MCs, CD341 endothelial cells, and CD341 hematopoietic progenitors on a background of stained nuclei. STRO-11 MCs appeared to preferentially localize to the periphery of the hematons (Figure 3D, Supporting Information Video S4). A large proportion of CD341hematopoietic cells significantly colocalized with MCs (i.e., 68% <10 mm from the nearest STRO-11cell). In comparison, approximately

only 15% of CD341cells were in close contact with endothelial cells, and a minority of CD341hematopoietic cells (<10%) were colocalized in the rare areas in which endothelial cells and STRO-11cells were in very close contact (Figure 3E–H).

Last, we further identified native MCs by the association of CD27117with STRO-1. There was STRO-1 and CD271 costaining in 486 9% of stromal cells (Pearson’s R 5 0.7) (Figure 3I), and specific subsets expressing either the STRO-1 or CD271 antigen were also identified. We observed that the CD2711/STRO-1-subset could form a vascular-looking structure, indicating pericyte-type cells. All hematons tested contained MCs expressing the STRO-1 and/or CD271 antigens.

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Relationship between the different parameters

of hematons

Through a single-hematon analysis, we were able to show that the vast majority of them contain mature cells from all hematopoietic lineages and immature cells up to the LTC-IC stage; we were able to detect SRC in about 40% of the structures despite the model boundaries and the low number of transplanted cells (Figure 4A). Only the smaller structures lacked the different levels of cell hierarchy (evaluated threshold of around 143 103cells; Supporting Information Figure S7).

In this study, we performed a statistical analysis of 297 hematons from 19 donors, taking into account intra- and inter-donor variability.

F I G U R E 2 (Continued)

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F I G U R E 3 Hematons contain functional mesenchymal cells that preferentially colocate with CD341cells. (A) Each hematon (4–10) from seven BM was dissociated and seeded in CFU-F assay medium. Most cell aggregates contained mesenchymal progenitors (means as bars). (B) After expansion, each single hematon-derived mesenchymal cell suspension from three BM samples was plated as a confluent monolayer and seeded by limiting dilution with control immunoselected CD341from healthy donors. We detected secondary progenitors in most cases. (C) Using the Poisson statistical model, we calculated the frequencies of LTC-IC evaluating the sustaining hematopoiesis properties of hematon-derived mesenchymal cells. (D) Visualization of the global structure confirmed the organization of hematons, with preferential localization of STRO-11cells on the periphery (Representative example (HD5) of hematons from healthy donors). Areas a, b, and c are shown in details. (a) In this area, we observed a vascular-type structure: capillaries/arterioles consisting of CD341endothelial cells bordered sometimes with STRO-11cells. (b) This area is rich in CD341cells, whose morphology evokes hematopoietic cells; most of these cells are localized with STRO-11cells (arrows). (c) This area includes CD341cells; most of these cells showed endothelial morphologies (arrows) and rarely colocated with STRO-11cells; white bars5 50 mm. (E) The distance between each CD341cell with hematopoietic morphology and the closest STRO-11cell (in a tri-dimensional plane) was measured in 1–3 hematons (out of at least 100 CD341cells) from three different BM samples. The same procedure was used to measure the distance between (F) each CD341cell and endothelial cell, (G) each STRO-11 cells and endothelial cells, and (H) each CD341cell and the nearest colocalized couple of STRO-11cell and endothelial cell. (I) To further characterize primitive native stromal cells in vivo, several hematons were costained with Abs against STRO-1 and CD271. STRO-1 and CD271 costained half of stromal cells (486 9%; a), but also revealed specific subsets expressing either STRO-1 (b) or CD271 antigen (c). We observed that the CD2711/STRO-1-subset could form a structure with a vascular appearance, suggesting pericyte-type cells (d). The

results are expressed as the mean6 ET. A: Blue, nucleus (Hoechst); red, CD341; green: STRO-11. F: blue, nucleus (Hoechst); red, CD2711; green: STRO-11[Color figure can be viewed at wileyonlinelibrary.com]

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We were able to analyze the results of the cell count (n5 250), including CD341(n5 19), GM (n 5 70), BFU-E (n 5 70), CFU-MK (n5 28), LTC-IC (n 5 86), and CFU-F (n 5 69), from single hema-ton. Using this analysis strategy, the“donor effect” was assessed at 28, 22, 68, 85, 37, 91, and 36%. Despite this variability, a positive correlation was observed between the main parameters, particularly clonogenic progenitors, but surprisingly, we did not find a correla-tion between CD341 cells and clonogenic progenitors or LTC-ICs (Figure 4B).

Then, we evaluated the influence of age and gender. Samples from males contained hematons enriched in CD341 cells and committed clonogenic progenitors, but samples from females contained hematons enriched in LTC-ICs (Figure 4C).

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D I S C U S S I O N

The results of this study showed that each BM-derived hematon was organized as a coherent multi-cellular aggregate with a lobular

F I G U R E 4 Relationships between variables. Relationships between variables were assessed using correlation coefficients and are represented graphically with a color-coded heatmap. Results concerning comparisons between patient gender are expressed as effect-sizes (standardized mean difference) and 95% confidence intervals and were compared to Cohen’s recommendations (Statistical power analysis for the behavioral sciences (2nd Ed.). New Jersey: Lawrence Erlbaum, 1988), who defined effect-size bounds as follows: small (ES: 0.2), medium (ES: 0.5), and large (ES: 0.8,“grossly perceptible and therefore large”) [Color figure can be viewed at wileyonlinelibrary.com]

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organization. Each structure appeared to contain most BM compo-nents: hematopoietic cells, adipocytes, MCs, and endothelial cells, organized in thin vessels or even arterioles and bone tissue. The rela-tive proportions of precursors from different hematopoietic lineages were approximately similar in hematons from the same BM and equiva-lent to the blended BM smears taken at diagnosis. This relationship may be related to the crush technique used in our laboratory because of its limited blood dilution effect.18Thus, each structure appeared to

not be dedicated to a preferred lineage but contained cells representa-tive of all lineages and could be considered to be representarepresenta-tive of BM organization.

We next evaluated the hematopoietic cell hierarchy in single hem-atons. The previously reported CD341cell enrichment in the hematon compartment6,8was due to a 2–3-fold higher percentage in each hem-aton compared to in the filtered cell suspension. At the progenitor level, no hematon was found to contain a preferential lineage, and all myeloid lineages were detected in most structures with a slight pre-dominance of the granulocyte/monocyte lineage. We observed a cor-relation with the number of cells, meaning that only the very small structures were practically devoid of progenitors.

Similarly, LTC-ICs, immature progenitors, were detected in more than 90% of hematons, sometimes at a high number, which was surpris-ing with regard to the number of CD341cells; this result is consistent with the higher frequency of LTC-ICs in the population of Lin-CD341/ CD901 cells localized in these structures.8 Surprisingly, we did not

observe a correlation between the number of CD341cells and that of LTC-ICs. This observation could be due to the difficulty in identifying CD341cells and LTC-ICs from the same hematon but could also indi-cate that the proportion of LTC-ICs may depend on other factors in these structures and not exclusively depend on the CD341cell content.

Because LTC-ICs are considered an in vitro equivalent of primitive hematopoietic cells,13these data prompted us to test cell suspensions

from single and mixed hematons to determine their ability to engraft in NSG mice by the intra-femoral route.15,19Our cut-off for the detection of human CD451cells in BM xenografts was low compared to previ-ous works, which was related to the intrinsic lower in vivo hematopoi-etic reconstitution potential of the adult tissue origin than the fetal origin or cord blood-derived HSCs.20In vivo T cell development origi-nating from hematons has been rarely achieved probably due to the use of NSG mice that were not engineered to study human thymopoie-sis16 and/or to the heterogeneity of the hematons. Nevertheless,

approximately half of the tested hematons generated human chimerism and exhibited human hematopoietic lympho-myeloid cells, suggesting that the frequency of SCID-repopulating cells (SRCs) was relatively high considering the approximately 10-fold lower total number of transplanted CD341cells compared to the number typically used from adult BM.15Moreover, despite the higher efficacy of the intra-bone

versus intravenous injection route,19,21a limited number of cells may

remain at the injection site,22particularly when low numbers of cells,

such as in hematons, need to be tested. Thus, we may have underesti-mated the numbers of hematon-derived HSCs using this model.

The presence of SRCs/HSCs and LTC-ICs strongly suggests the presence of hematopoietic niches in most hematons. However, we

detected LTC-ICs in nearly all hematons, but only half of the hematons engrafted in NSG mice, suggesting a greater heterogeneity of hema-tons as a shelter for HSCs and/or LTC-ICs, even if the likely less imma-ture LTC-ICs could only be maintained in a complex niche.13 This

discrepancy and the fact that the BM richest in LTC-ICs did not pro-duce multilineage chimerism indicates a certain degree of commitment of LTC-ICs compared with stem cells, confirming that LTC-ICs and SRC/HSCs overlap but are not identical.23

The use of a 3.6-fold more-sensitive mouse model than the NOD/ SCID mouse thus facilitates the detection of HSCs,24albeit numerous

studies have demonstrated the role of the human microenvironment in xeno-engraftment, and several groups are currently improving mouse models through “humanization” strategies.15,25 Here, we cannot exclude the role of MCs in the engraftment of HSCs.26,27Indeed, in most hematons, we detected CFU-Fs, and most single hematon-derived stromas could sustain hematopoiesis in vitro. To identify MCs likely to improve engraftment in vivo,28we evaluated the expression of

STRO-1, a marker of native primary cells29,30 with high-growth

capacity.31,32 We confirmed this result by costaining the cells with

anti-CD271, which is known to identify a CFU-F-enriched mesenchy-mal subset.17,33This never previously reported in vivo costaining

dem-onstrates that most MCs were costained by the two antibodies and that a subset of CD2711STRO-cells could correspond to pericytes.34

Electronic and confocal microscopy results showed that MCs were preferentially located as a network at the periphery of hematons. This observation, together with the observations that hematons dissociated when MCs adhered to the culture plate and that the disruption of BM hematons induced by some leukemias was related to a marked decrease in mesenchymal progenitors,7,35suggest that these cells are critical for supporting cell cohesion, an in vivo role that has not been previously described. Thus, we coinjected mice with MCs likely to con-tribute to engraftment through the“humanization” of NSG mice.

Furthermore, most CD341 cells were colocalized with STRO-11 MCs, whereas a limited number of CD341cells were located close to endothelial cells or endothelial/STRO-11cells. Further studies are nec-essary to better identify hematopoietic CD341subsets in these differ-ent locations, but this observation indicates the presence of functional niches in hematons.

We observed intra- and inter-donor variabilities in the data, sug-gesting an individual functional efficiency of human BM elementary units that was partially explained by the influence of age and gender. This observation suggests a genetic determinism of BM tissue, which is a seldom explored concept that should be further evaluated. However, taking into account these variabilities, we detected a correlation between the number of cells (hematon size) and clonogenic progeni-tors. However, no correlation was observed between the CD341 cell content and progenitors, in particular the LTC-ICs, indicating the limits of this marker in these structures.

In recent years, several types of niches have been described in mice.4,36 The endosteal niche has been suggested as the major BM

microdomain ensuring HSC maintenance, but recently, the vascular niches have been proposed to be the major candidates for stromal

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factors in host HSCs, megakaryocytes, endothelial cells, and pericytes.37–40Here, the detection of blood vessels, MCs, and osseous tissues supports the presence of most of categories of niches.

Our results demonstrate that human hematons constitute BM tissue subunits containing all the cellular actors of the hematopoietic niche and that they are easy to collect and represent a unique model for under-standing the interdependence between immature hematopoietic cells and the BM microenvironment. Access to human hematons provides a foundation for novel investigations such as the following: (i) to evaluate the limits of extrapolation from data obtained with mouse models,16,26,41–46 ii) to advantageously replace the ex vivo artificially reconstructed 3D models of the BM microenvironment,47–53(iii) to evalu-ate the application of these structures in cell therapy and to study mecha-nisms such as cell egress,26,54(iv) to produce ex vivo expansion conditions

when self-renewal is difficult to reproduce,55,56and (v) to study

oncogen-esis and/or the expansion of initiating leukemia stem cells and/or the relationship between malignant clones and the microenvironment.57–60

Hematons represent a promising model of human hematopoiesis and open new research approaches, which are necessary for the develop-ment of new therapeutic strategies adapted to in vivo cellular behavior.61

A C K N O W L E D G M E N T S

The authors thank Dr. Chantal Rapatel, head of the flow cytometry platform (CHU Estaing, Clermont-Ferrand), Prof. Salvatore Valitutti for welcoming AJ to INSERM U563 (Dynamique moleculaire des interactions lymphocytaires, Toulouse) for confocal microscopy train-ing. They also thank Sandrine Saugues and Fanny Soule (Centre de Ressources Biologiques (CRB) Auvergne) for cryopreserving biologi-cal samples, Dr. Jean-Louis Couderc, Caroline Vachias (microscopy confocal platform), Christelle Blavignac and Claire Szczepaniak (Centre d’Imagerie Cellulaire Sante Universited’Auvergne for elec-tronic microscopy). They are indebted to Aurelie Briançon et Char-lène Fernandes for expert technical assistance and for GMP preparation of cell culture medium and to Julien Tilliet (IRCM animal core facility) for technical assistance during in vivo assays. They thank Dominique Chadeyron for manuscript preparation.

C O N F L I C T O F I N T E R E S T S No conflict of interest to declare.

A U T H O R C O N T R I B U T I O N S

Project leader, designed and supervised the confocal microscopy procedure, contributed to all aspects of the work and wrote the manuscript: Alexandre Janel

Project leader for long-term culture assays and their interpreta-tion; engineer of CRB Auvergne: Juliette Berger

Project leader for flow cytometry analysis and cell sorting: Celine Bourgne

Project leader for cell culture assays and participated in writing the manuscript: Nathalie Boiret-Dupre

Determined cell number at the cell aggregate level (Masters 1 student): Richard Lemal

Participated in cytological cell identification and dissociation pro-cedures: Frederique Dubois-Galopin

Project leader for immunohistochemical analysis: Pierre Dechelotte Participated in immunohistochemical analysis: Charlotte Bothorel Participated in transplantation experiments, analysed data (PhD student, LSHL): Sara Chabi

Experts in HSC transplantation, they collected the BM filters from donors: Eric Hermet and Jacques-Olivier Bay

Carried out and/or supervised the statistical analyzes: Bruno Pereira and Celine Lambert

Designed and conducted the in vivo transplantation assays, ana-lysed data: Françoise Pflumio and Rima Haddad

Head of the laboratory, designed and supervised the overall research, and wrote the manuscript: Marc G. Berger

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S U P P O R T I N G I N F O R M A T I O N

Additional Supporting Information may be found online in the sup-porting information tab for this article.

How to cite this article: Janel A, Berger J, Bourgne C, et al. Bone marrow hematons: An access point to the human hemato-poietic niche. Am J Hematol. 2017;92:1020–1031. https://doi. org/10.1002/ajh.24830

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