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Osteoclasts Are Not Crucial for Hematopoietic Stem Cell Maintenance in Adult Mice

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Osteoclasts Are Not Crucial for Hematopoietic Stem Cell Maintenance in Adult Mice ARTICLES Hematopoiesis & Hematopoietic Stem Cell Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice Carmen Flores,1 Ilana Moscatelli,1 Christian S. Thudium,1,2 Natasja Stæhr Gudmann,2 Jesper S. Thomsen,3 Annemarie Brüel,3 Morten A. Karsdal,2 Kim Henriksen,2 and Johan Richter1 1Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Sweden; 2Nordic Bioscience, Herlev, Denmark; and 3Department of Biomedicine-Anatomy, Aarhus University, Aarhus, Denmark ABSTRACT The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplant- ed hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclas- togenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell func- tion by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97-98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation2013 was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knock-out recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice. Introduction mediated resorption promotes mobilization of HSCs and pro- genitors from the niche to the circulation by cathepsin K- The osteoclast responsible for the resorption of bone and mediated cleavage of CXCL12.18 In contrast to this, osteoclast the osteoblast ensuring formation of new bone are two Foundationinhibition was also shown to increase mobilization.19,20 In unique cell types that continuously repair and maintain the addition, it has been demonstrated that mice lacking calcium- human skeleton through a tightly co-ordinated process sensing receptors have reduced numbers of HSCs in the BM, known as bone remodeling. During ontogeny, both osteo- indicating that the calcium released as a consequence of bone clasts and osteoblasts are essential for the formationStorti of the resorption is important for the correct localization of HSCs specialized microenvironmental niche where the blood-form- and that this is specified by calcium-sensing receptors.21,22 ing hematopoietic stem cells reside, the hematopoietic Furthermore, when normal mice were treated with the bis- niche.1,2 The hematopoietic stem cells (HSCs) interaction with phosphonate alendronate (that inhibits and induces apoptosis their microenvironment is critical when maintaining normal in osteoclasts), a slight reduction of HSCs in the BM was hematopoiesis and their specific fate is determined through observed.23 complex, bidirectional interactions with various cell types In the present study, aiming to explore the role of the osteo- and stromal cell components.3-5 In the adult bone marrow clast for maintenance of adult hematopoiesis, two (BM), different stromal cells regulate©Ferrata HSCs. Osteoblasts main- osteopetrotic mouse models were used: the oc/oc and RANK tain the HSCs in an undifferentiated, quiescent state by pro- KO. Oc/oc mice with a mutation in the Tcirg1 gene lack osteo- viding inhibitory signals like Jagged and Angiopoietin 1, but clastic V-ATPase activity and their resorptive function has also by expressing N-cadherin and VCAM that interact with been completely abolished, but they do have a large number integrins expressed on HSCs, attaching them to the niche.6-11 of osteoclasts and a severe osteopetrotic phenotype with a Vascular stromal cells, e.g. sinusoidal endothelial cells,12 short life expectancy of 3-4 weeks.24 In contrast, the RANK fibroblast-like reticular cells and Nestin+ mesenchymal stem KO mouse is defective in osteoclast differentiation and is, cells that express high levels of SDF-1/CXCL12 also play key therefore, devoid of osteoclasts. Both models suffer from roles in HSC maintenance.13-17 osteopetrosis, but the phenotype is less severe and the life Lately, several reports have highlighted the importance of expectancy is longer in the RANK KO than in the oc/oc the osteoclast in regulation of the hematopoietic niche, but its mouse.25 By irradiating wild-type mice, and subsequently precise role for this process under various conditions still transplanting fetal liver cells from either oc/oc or RANK KO remains controversial. It has been shown that osteoclast- mice, we generated adult mice with osteopetrosis suitable for ©2013 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2013.089466 The online version of this article has a Supplementary Appendix. Manuscript received on April 9, 2013. Manuscript accepted on October 1, 2013. Correspondence: [email protected] 1848 haematologica | 2013; 98(12) Osteoclasts and HSC maintenance studying the role of osteoclasts for maintenance of Splenocyte osteoclast differentiation and resorption hematopoiesis in this setting. Splenocytes were isolated as previously described30 and were differentiated into mature osteoclasts (Online Supplementary Methods). Resorption pits were visualized by staining with Methods Mayer's hematoxylin. The resorbed area was measured using NewCAST software (Visiopharm, Hørsholm, Denmark) with an Mice Olympus IX-70 microscope (Olympus, Center Valley, PA, USA). Breeding pairs of oc/+ mice (CD45.2)26 and B6SJL (CD45.1) were obtained from the Jackson Laboratory (Bar Harbor, ME, TRAP staining of osteoclasts USA). RANK+/− mice (CD45.2) were obtained from Amgen Fixed osteoclasts in cell culture were stained using the (Seattle, WA, USA).25 All experiments were performed according Leukocyte TRAP staining kit (Sigma-Aldrich). Histological sec- to protocols approved by the local animal ethics committee (num- tions of bone were stained for TRAP, counterstained with Mayer’s ber 333-11). hematoxylin, and mounted with DPX. Genotyping of mice Biomarkers Mice were genotyped by PCR of tail tips, as described previ- TRAP5b and CTX-I were measured in serum using mouse ously.27 TRAP5b ELISA kit and RatLaps ELISA kit (Immunodiagnostics Systems), respectively, according to standard protocols. Fetal liver cell harvesting Fetal liver cells were collected as described previously.28 Statistical analysis Statistical analysis was performed either with unpaired two- Primary transplantations sided Student’s t-test, one-way ANOVA, Bonferroni or Mann- Three-month old B6SJL (CD45.1) recipients were transplanted Whitney. *P<0.05; **P<0.01; ***P<0.001. with 2 million freshly thawed FL cells (CD45.2) administered by 2013 tail vein injection after lethal irradiation (950 cGy). Post transplant mice were treated with ciprofloxacin. Results Secondary transplantations Transplantation of oc/oc and RANK KO HSCs to A total of 2 x 105 BM cells (CD45.2) were harvested from pri- wild-type mice leads to mild osteoclast-rich and mary recipients and transplanted into secondary recipients 5 osteoclast-poor osteopetrosis in recipients, (CD45.1) in a competitive setting with 3 x 10 wild-type BM cells respectively (CD45.1/2). Recipient wild-type mice were lethally irradiated and Foundationsubsequently transplanted with FL cells from oc/oc, RANK Flow cytometry analysis of peripheral blood, bone KO or littermate control mice. The mice transplanted with marrow and fetal liver cells FL cells from oc/oc and RANK KO will be referred to as For engraftment, cells were stained with Ly5.1-PeCy5 and oc/oc recipients (oc/oc-R) and RANK KO recipients Ly5.2-FITC. For lineage analysis and stem cell analysis, cells were (RANK KO-R), respectively, and mice that received WT stained with a cocktail of antibodies directed against Stortilineage mark- (+/+) or heterozygous oc/+ or RANK +/- FL cells will be ers, Sca1, c-Kit and SLAM-markers, as described in the Online referred to as control recipients (Ctrl-R). When the fre- Supplementary Methods. quency of LSK-SLAM HSCs and the cell cycle status in the LSK population were analyzed in the three donor cell pop- Cell cycle analysis ulations, no difference was observed (Online Supplementary For the cell cycle analysis of BM, antibodies were directed Figure S1A and B). Six weeks post transplantation the per- against lineage-positive cells (CD4, CD5, GR1, Lyt2, Ter119, centage of donor cells in PB reached 97-98% in all groups, CD11b and CD45R/b220)-PeCy5, c-kit-APC780, Sca1-APC, and this level of engraftment was sustained at the time of CD48-FITC, CD150-PeCy7, ©FerrataKi67-PE and DAPI-Pacific Blue. For termination, 15 weeks after transplantation, in both PB the analysis of FL cells, the lineage cocktail was (CD3, Ter119, and BM. B220, Gr1)-PECy5 (Becton
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