Human, mouse, and dog bone marrow show similar mesenchymal stromal cells within a distinctive microenvironment
2021; Elsevier BV; Volume: 100; Linguagem: Inglês
10.1016/j.exphem.2021.06.006
ISSN1873-2399
AutoresBerenice Meza-León, Dita Gratzinger, Alicia G. Aguilar-Navarro, Fany G. Juárez-Aguilar, Vivienne I. Rebel, Emina Torlakovic, Louise E. Purton, Elisa Dorantes‐Acosta, Argelia Escobar‐Sánchez, John E. Dick, Eugenia Flores‐Figueroa,
Tópico(s)Cancer Cells and Metastasis
Resumo•Human bone marrow (BM) histology is more similar to dog than mouse BM histology.•Mouse BM has higher cellularity and megakaryocyte density compared with human and dog BM.•The frequency of LepR+ mouse BMSCs is similar frequency to those of human and dog CD271+ BMSCs.•BMSC density does not change with aging in human, dog, or mouse bone marrow. Bone marrow stromal cells (BMSCs) are a key part of the hematopoietic niche. Mouse and human BMSCs are recognized by different markers (LepR and NGFR/CD271, respectively). However, there has not been a detailed in situ comparison of both populations within the hematopoietic microenvironment. Moreover, dog BMSCs have not been characterized in situ by any of those markers. We conducted a systematic histopathological comparison of mouse, human, and dog BMSCs within their bone marrow architecture and microenvironment. Human and dog CD271+ BMSCs had a morphology, frequency, and distribution within trabecular bone marrow similar to those of mouse LepR+ BMSCs. However, mouse bone marrow had higher cellularity and megakaryocyte content. In conclusion, highly comparable bone marrow mesenchymal stromal cell distribution among the three species establishes the validity of using mouse and dog as a surrogate experimental model of hematopoietic stem cell–BMSC interactions. However, the distinct differences in adipocyte and megakaryocyte microenvironment content of mouse bone marrow and how they might influence hematopoietic stem cell interactions as compared with humans require further study. Bone marrow stromal cells (BMSCs) are a key part of the hematopoietic niche. Mouse and human BMSCs are recognized by different markers (LepR and NGFR/CD271, respectively). However, there has not been a detailed in situ comparison of both populations within the hematopoietic microenvironment. Moreover, dog BMSCs have not been characterized in situ by any of those markers. We conducted a systematic histopathological comparison of mouse, human, and dog BMSCs within their bone marrow architecture and microenvironment. Human and dog CD271+ BMSCs had a morphology, frequency, and distribution within trabecular bone marrow similar to those of mouse LepR+ BMSCs. However, mouse bone marrow had higher cellularity and megakaryocyte content. In conclusion, highly comparable bone marrow mesenchymal stromal cell distribution among the three species establishes the validity of using mouse and dog as a surrogate experimental model of hematopoietic stem cell–BMSC interactions. However, the distinct differences in adipocyte and megakaryocyte microenvironment content of mouse bone marrow and how they might influence hematopoietic stem cell interactions as compared with humans require further study. Mouse models have been widely used to study hematopoiesis, including the in situ location of hematopoietic stem cells (HSCs) and their progeny. Unfortunately, there is little direct information on the in situ human bone marrow microenvironment, including the location of hematopoietic stem/progenitor cells (HSPCs) [1Aguilar-Navarro AG Meza-León B Gratzinger D et al.Human aging alters the spatial organization between CD34+ hematopoietic cells and adipocytes in bone marrow.Stem Cell Rep. 2020; 15: 317-325Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 2Corselli M Chin CJ Parekh C et al.Perivascular support of human hematopoietic stem/progenitor cells.Blood. 2013; 121: 2891-2901Crossref PubMed Scopus (134) Google Scholar, 3Flores-Figueroa E Varma S Montgomery K Greenberg PL Gratzinger D Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow.Lab Invest. 2012; 92: 1330-1341Crossref PubMed Scopus (59) Google Scholar, 4Gomariz A Isringhausen S Helbling PM Nombela-Arrieta C Imaging and spatial analysis of hematopoietic stem cell niches.Ann NY Acad Sci. 2020; 1466: 5-16Crossref PubMed Scopus (9) Google Scholar, 5Sacchetti B Funari A Michienzi S et al.Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment.Cell. 2007; 131: 324-336Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar, 6Tormin A Li O Brune JC et al.CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization.Blood. 2011; 117: 5067-5077Crossref PubMed Scopus (297) Google Scholar], so the findings using mouse models have been implicitly extrapolated to the human setting. However, in most mouse strains, lymphocytes represent about 70%–80% of all circulating leukocytes, and neutrophils represent only 20%–30% of circulating cells [7Mestas J Hughes CCW Of mice and not men: differences between mouse and human immunology.J Immunol. 2004; 172: 2731-2738Crossref PubMed Scopus (2128) Google Scholar,8O'Connell KE Mikkola AM Stepanek AM et al.Practical murine hematopathology: a comparative review and implications for research.Comp Med. 2015; 65: 96-113PubMed Google Scholar]. Cows, sheep, and goats are other mammal species that also have a predominantly lymphoid-biased hematopoiesis [9Mohammed A Campbell M Youssef FG Serum copper and haematological values of sheep of different physiological stages in the dry and wet seasons of Central Trinidad.Vet Med Int. 2014; 2014e972074Crossref PubMed Scopus (15) Google Scholar,10Roland L Drillich M Iwersen M Hematology as a diagnostic tool in bovine medicine.J Vet Diagn Invest. 2014; 26: 592-598Crossref PubMed Scopus (131) Google Scholar]. In contrast, neutrophils represent 60%–65% of circulating leukocytes in humans and dogs, and their bone marrow has a higher percentage of myeloid cells [7Mestas J Hughes CCW Of mice and not men: differences between mouse and human immunology.J Immunol. 2004; 172: 2731-2738Crossref PubMed Scopus (2128) Google Scholar,11Tvedten HW Hematology of the normal dog and cat.Vet Clin North Am Small Anim Pract. 1981; 11: 209-217Crossref PubMed Scopus (16) Google Scholar]. It is well established that HSCs and progenitor cells are regulated by their interactions within specific microenvironmental niches; B lymphopoiesis is regulated by osteoblasts [12Ding L Morrison SJ Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches.Nature. 2013; 495: 231-235Crossref PubMed Scopus (750) Google Scholar] and specific subpopulations of mesenchymal stromal cells (BMSCs) in humans [13Torlakovic E Tenstad E Funderud S Rian E CD10+ stromal cells form B-lymphocyte maturation niches in the human bone marrow.J Pathol. 2005; 205: 311-317Crossref PubMed Scopus (17) Google Scholar] and in mice, where they have a peri-arteriolar location [14Shen B Tasdogan A Ubellacker JM et al.A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis.Nature. 2021; 591: 438-444Crossref PubMed Scopus (34) Google Scholar]. On the other hand, myelopoiesis is regulated by adipocytes [15Boyd AL Reid JC Salci KR et al.Acute myeloid leukaemia disrupts endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow niche.Nat Cell Biol. 2017; 19: 1336-1347Crossref PubMed Scopus (91) Google Scholar,16Zhang Z Huang Z Ong B Sahu C Zeng H Ruan HB Bone marrow adipose tissue-derived stem cell factor mediates metabolic regulation of hematopoiesis.Haematologica. 2019; 104: 1731-1743Crossref PubMed Scopus (18) Google Scholar] and BMSCs. BMSCs (reviewed by Wei and Frenette [17Wei Q Frenette PS Niches for hematopoietic stem cells and their progeny.Immunity. 2018; 48: 632-648Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar] and Crane et al [18Crane GM Jeffery E Morrison SJ Adult haematopoietic stem cell niches.Nat Rev Immunol. 2017; 17: 573-590Crossref PubMed Scopus (330) Google Scholar]), megakaryocytes, and endothelial cells are key microenvironmental cells for HSCs and HSPCs. The differences between lymphoid and myeloid bias in mouse versus human and dog hematopoiesis suggest that the bone marrow microenvironments of dogs and humans are more similar than those of humans and mice. However, a detailed direct comparison of bone marrow stromal cell immunomorphology and bone marrow architecture between mouse (C57BL/6 and SCID, two of the most widely used mouse strains), human, and dog trabecular bone marrow has not been conducted. The dog is the classic model of human stem cell transplantation [19Graves SS Rezvani A Sale G et al.A canine model of chronic graft-versus-host disease.Biol Blood Marrow Transplant. 2017; 23: 420-427Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar] and also serves as a model in which to study many human hematopoietic diseases [20Hoffman AM Dow SW Concise review: stem cell trials using companion animal disease models.Stem Cells. 2016; 34: 1709-1729Crossref PubMed Scopus (85) Google Scholar,21Hoffman JM Creevy KE Franks A O'Neill DG Promislow DEL The companion dog as a model for human aging and mortality.Aging Cell. 2018; 17: e12737Crossref PubMed Scopus (40) Google Scholar]. However, the differences between the dog hematopoietic microenvironment and those of humans and mice have not been explored thoroughly. Hence, a fundamental question remains: How relevant are the mouse and dog microenvironments to the human microenvironment? A systematic comparison between the three species is needed. Pediatric human bone marrow biopsy specimens (n = 3), obtained from the pathology archive of the Hospital Infantil de México Federico Gómez, comprised iliac crest core biopsy specimens from oncology patients aged <16 years with no marrow involvement (HIM/2013/034). Adult human bone marrow samples (n = 9) were obtained from patients undergoing hip replacement surgery (arthrosis and fractures as underlying etiology) at the Instituto Mexicano del Seguro Social. The intertrabecular bone marrow spaces examined were distant from the articular surface and did not manifest histologic changes associated with degenerative joint disease, such as cystic degeneration and overlying cartilage defects. Samples consisted of hematopoietically active femoral necks of hematologically healthy patients aged 53–92 years; oral consent was obtained (R-2012-785-092). Mouse sternums from 6-week-old (n = 5) and 24-month-old (n = 4) C57Bl6 mice were provided from banked formalin-fixed paraffin-embedded (FFPE) blocks from the University of Texas Health Science Center at San Antonio (UTHSCSA, San Antonio, TX). Sternums from 15-week-old severe combined immunodeficient (SCID) mice (n = 5) were obtained from the University Health Network (UHN) animal facility (Toronto, ON, Canada). Dog bone marrow samples (mixed breed, n = 8; Siberian Husky, n = 1; and Pomeranian, n = 1) were obtained from haematopoietically active femoral necks of animals aged 3 months to 8 years undergoing excision of the femoral head in private animal hospitals in Mexico City. All experimental protocols using dog samples were reviewed and approved by the Institutional Subcommittee for Experimental Animal Care (SICUAE) of the Universidad Nacional Autónoma de México (UNAM). All samples were scored by three independent hematopathologists to confirm normal active hematopoietic sites. Mouse sternums were cleaned of excess muscle tissue and fixed in 4% paraformaldehyde solution (Sigma P6148) overnight at 4°C. Human and dog bones were cut into 5-cm-thick blocks to be fixed in 10% formaldehyde solution (Meyer 1405-4000) at room temperature for 24 hours. After fixation, human and dog bones were cut into 5-mm slices and placed in 12% Ethylenediamine tetraacetic acid (EDTA) (Sigma 5134) in phosphate-buffered saline (PBS) solution, pH 8, for decalcification. The solution was changed daily for 4 to 5 days (mouse) and 15 days (human and dog). After decalcification, samples were rinsed for 30 minutes under tap water and placed in 70% ethanol. Samples were dehydrated through a series of graded ethanol solutions and then embedded in paraffin using a semiautomatic system (KOS, Milestone Medical, Kalamazoo, MI). Hematoxylin and eosin (H&E)–stained histologic sections were reviewed by three independent hematopathologists; cellularity was evaluated using the percentage of hematopoietic cells in relation to the percentage of mature adipocytes in each intertrabecular space. Myeloid-to-erythroid (M:E) ratio was estimated in intertrabecular spaces away from cortical bone. The number of megakaryocytes was quantified in an area of 1 mm2, equivalent to two intertrabecular spaces. Immunohistochemistry (IHC) was performed on 4-µm tissue sections, which were placed on electro-charged slides and deparaffinized overnight at 60°C. Slides were placed twice in Ottix (Catalog No. X0076, Diapath, Martinengo, Lombardy, Italy) for 8 minutes, then in Ottix shaper (Catalog No. X0096, Diapath) twice for 5 minutes, and then in distilled water. Antigen retrieval was performed using citrate buffer solution, pH 6 (Catalog No. S1804, Sigma-Aldrich, St. Louis, MO), in a microwave multifunctional tissue processor (KOS, Milestone). Endogenous peroxidase activity was blocked for 20 minutes with a ready-to-use commercial solution (Vector SP-6000). Normal horse serum (2.5%, Vector S-2012) was used for antigen blocking. Serum was decanted, and slides were incubated with primary antibody overnight at 4°C. The following antibodies were used: rabbit anti-CD271 clone EP1039Y (Catalog No. NU522-UC, BioGenex, Fremont, CA), diluted 1:50, and goat anti-LepR (Catalog No. AF-497, R&D Systems, Minneapolis, MN), diluted 1:250. Antibodies were diluted with a ready-to-use low-background diluent (Catalog No. K004, Diagnostic Biosystems, Pleasanton, CA). Slides were washed in PBS–Tween (0.1%) and incubated for 30 minutes at room temperature with secondary antibody (HRP Kit polymer, vector anti-rabbit MP-7401; anti-goat MP-7405, Vector Laboratories, Burlingame, CA). Slides were washed in PBS–Tween (0.1%) after the incubation period and incubated between 5 and 10 minutes with diaminobenzidine solution (Vector, SK-4103) and counterstained with hematoxylin. Slides were scanned and digitized using an Aperio CS2 scanner (Leica Biosystems, Buffalo Grove, IL) using the objective 20 × /0.75 NA Plan Apo at 21°C. Images were stored using Aperio ImageScope (Version 12.3.2.8013). Image analysis was performed with the open-source software CellProfiler 2.1.1 [22Lamprecht MR Sabatini DM Carpenter AE CellProfiler: free, versatile software for automated biological image analysis.Biotechniques. 2007; 42: 71-75Crossref PubMed Scopus (584) Google Scholar]. A previously published pipeline was used for the quantification of BMSCs [23Johnson RC Kurzer JH Greenberg PL Gratzinger D Mesenchymal stromal cell density is increased in higher grade myelodysplastic syndromes and independently predicts survival.Am J Clin Pathol. 2014; 142: 795-802Crossref PubMed Scopus (11) Google Scholar], with slight modifications made for each marker. We quantified the area of positivity of each marker according to the total area in a 20 × magnification field. Statistical analysis was performed with GraphPad Prism 5 software (GraphPad, San Diego, CA); nonparametric tests for comparisons including Kruskal-Wallis, Mann-Whitney, and Spearman correlations were used. Trabecular bone specimens were selected from the three species. Bone marrow histologic sections from human iliac crests or femoral necks, mouse sternum, and dog femoral necks revealed an extensive trabecular bone meshwork (Figure 1A–D). Mouse bone marrow had the highest hematopoietic cellularity (100%) for both strains examined (Figures 1B,C and 2A). The high cellularity in mouse bone marrow correlates with the absence of adipocytes. Both human and dog bone marrow had a significantly lower cellularity than mouse bone marrow (median [MED] = 45%, interquartile range (IQR) = 27.5%, and MED = 70%, IQR = 56.2%, respectively) (p < 0.0001) (Figures 1A–D and 2A). Megakaryocyte content was higher in SCID than in C57BL/6 mice (MED = 83/mm2 and 29/mm2, IQR = 33/mm2 and 24/mm2, respectively) (p = 0.0079) (Figures 1F,G and 2B). Both mouse strains had a higher megakaryocyte content than human and dog bone marrow (MED = 6.75/mm2, IQR = 3.5/mm2, and MED= 7.0/mm2, IQR = 4.2/mm2, respectively) (p < 0.0001) (Figures 1E–H and 2B). Megakaryocyte content was higher in 6-week than 24-month C57BL/6 mice (p = 0.0159). There was a significant difference in the M:E ratio among the species (p = 0.0138). SCID mice had the lowest M:E ratio (Figures 1K and 2C). Samples from 6-week-old C57BL/6 mice had a lower M:E ratio compared with 24-month-old mice (p = 0.0179) (Figures 1J and 2C). Cellularity varied more among human and dog bone marrow samples than among those of mice.Figure 2Quantitative evaluation of human, mouse, and dog bone marrow. (A) Percentage of marrow hematopoietic cellularity across species. (B) Number of megakaryocytes per square millimeter across species. (C) M:E ratio across species. Kruskal-Wallis test. (A–C) Human (n = 12), mouse (n = 9), and dog (n = 10) bone marrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In human bone marrow, as previously described [6Tormin A Li O Brune JC et al.CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization.Blood. 2011; 117: 5067-5077Crossref PubMed Scopus (297) Google Scholar,24Cattoretti G Schiró R Orazi A Soligo D Colombo MP Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro.Blood. 1993; 81: 1726-1738Crossref PubMed Google Scholar], CD271 antibody recognized delicate ramifying bone marrow mesenchymal stromal cells with reticular morphology compatible with BMSC morphology in situ (Figure 3A). A population of delicate ramifying cells morphologically similar to the human CD271+ population was also detected in dog bone marrow (Figure 3B). In both species, hematopoietic cells were negative for CD271 (Figure 3A,B). CD271+ BMSCs were sparsely distributed within the hematopoietic parenchyma, forming delicate simple branches extending between hematopoietic cells (Figure 3A,B). CD271+ BMSCs could also be found in close proximity to trabecular bone (Figure 3C,D). In perivascular sites, they were observed to form thin cell layers outlining sinusoidal endothelial cells (Figure 3E,F) and adjacent to megakaryocytes (Figure 3G,H). A thick and branching cell layer of CD271+ BMSCs was regularly identified to peripherally outline arterioles (Figure 3I,J). Given the marked predominance of sinusoidal vascular surface area over arteriolar vasculature and trabecular bone surface area in the marrow space, the presence of CD271+ BMSCs was much more extensive in perisinusoidal than in the periarteriolar or peritrabecular locations. However, the morphology of BMSCs around arterioles was marked by its density and prominent arborization, as compared with the single-cell layer lining and less prominent arborization around marrow sinusoids. Leptin receptor+ (LepR+) cells in mouse bone marrow, resemble in morphology and distribution, the CD271+ cells in human and dog bone marrow. The reticular morphology was more evident in LepR+ BMSCs in SCID mice than in those in C57BL/6 mice (Figure 4A,B). In mouse bone marrow, in both the CD57BL/6 and SCID strains, LepR+ cells also have a similar distribution. Just like CD271+ BMSCs in human and dog bone marrow, the LepR+ BMSCs were identified surrounding sinusoids (Figure 4E,F) and some perisinusoidal megakaryocytes (Figure 4G,H). LepR+ BMSCs in both mouse strains present the characteristic multilayer branching distribution of periarteriolar BMSCs (Figure 4I,J) observed in both dog and human specimens. Furthermore, murine LepR+ BMSCs were also distributed adjacent to bone and exhibited arborization into the hematopoietic parenchyma (Figure 4A–D). On the basis of image analysis using Cell Profiler software (Figure 5A), human and dog bone marrow had similar densities of CD271+ BMSCs (MED = 22.52, IQR = 9.51, and MED = 18.48, IQR = 7.17, respectively; p = 0.8567). Mouse bone marrow LepR+ BMSCs, from both strains C57BL/6 and SCID, were similar in density to human and dog CD271+ BMSCs (MED = 16.78, IQR = 3.11, and MED = 16.53, IQR = 5.96, respectively; p = 0.6993) (Figure 5B). We found no statistical differences in density between BMSCs from young samples (P = 0.1735) and those from old samples (P = 0.6710) in dogs, humans, or mice. We correlated age and marrow cellularity, M:E ratio, megakaryocyte number, and BMSC density across human, dog, and mouse bone marrow (C57BL/6). We found that human bone marrow and dog bone marrow undergo similar age-related changes, which include a decrease in bone marrow cellularity with aging and no changes in megakaryocyte content. In contrast, mouse bone marrow had no correlation with age and cellularity but had a strong negative correlation with the number of megakaryocytes. The M:E ratio had a strong positive correlation with age in mice and a lower correlation in humans and dogs. We found no significant correlation between age and density of BMSCs in humans, mice, or dogs (Figure 6). In contrast to human and dog bone marrow, mouse bone marrow has few adipocytes and higher cellularity in the trabecular bone, which does not decrease with age. Adipocytes are known negative regulators of lymphopoiesis [25Pang WW Price EA Sahoo D et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc Natl Acad Sci USA. 2011; 108: 20012-20017Crossref PubMed Scopus (460) Google Scholar]. Hence, the decreased number of adipocytes in mouse correlates with the increased number of lymphocytes in this species. Mouse bone marrow also had higher megakaryocyte density than human and dog trabecular bone marrow. Moreover, the hematopoietic compartment also differs between mouse strains: Two of the most frequently used mouse models, C57BL/6 and SCID, exhibited significant differences in megakaryocyte content. We report concordance between the distributions of immunophenotypically distinct but morphologically similar BMSCs between species. BMSC density was conserved with age and did not vary between the species analyzed, despite the differences in lymphoid and myeloid bias among them. CD271+ and LepR+ BMSCs have similar morphology, density, and distribution. However, both subpopulations may have transcriptomic differences to support the more myeloid/lymphoid bias hematopoiesis, respectively. Our data correlate with recent observations that the frequencies of mouse LepR+ BMSCs [14Shen B Tasdogan A Ubellacker JM et al.A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis.Nature. 2021; 591: 438-444Crossref PubMed Scopus (34) Google Scholar] and human CD271+ BMSCs [1Aguilar-Navarro AG Meza-León B Gratzinger D et al.Human aging alters the spatial organization between CD34+ hematopoietic cells and adipocytes in bone marrow.Stem Cell Rep. 2020; 15: 317-325Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar] did not change with aging. However, changes in the functional properties of BMSCs [26Ganguly P El-Jawhari JJ Burska AN Ponchel F Giannoudis PV Jones EA The analysis of in vivo aging in human bone marrow mesenchymal stromal cells using colony-forming unit-fibroblast assay and the CD45lowCD271+ phenotype.Stem Cells Int. 2019; 20195197983Crossref PubMed Scopus (39) Google Scholar] and in the subpopulation frequencies and their distribution with aging [27Maijenburg MW Kleijer M Vermeul K et al.The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.Haematologica. 2012; 97: 179-183Crossref PubMed Scopus (74) Google Scholar] have been reported. In larger human studies, it is well documented that adipocytes progressively replace hematopoietic areas in all anatomic locations with aging [28Hartsock RJ Smith EB Petty CS Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest: a study made from 177 cases of sudden death examined by necropsy.Am J Clin Pathol. 1965; 43: 326-331Crossref PubMed Scopus (220) Google Scholar, 29Nehlin JO Jafari A Tencerova M Kassem M Aging and lineage allocation changes of bone marrow skeletal (stromal) stem cells.Bone. 2019; 123: 265-273Crossref PubMed Scopus (15) Google Scholar, 30Travlos GS Normal structure, function, and histology of the bone marrow.Toxicol Pathol. 2006; 34: 548-565Crossref PubMed Scopus (195) Google Scholar]. In contrast, cellularity was not reduced in the sternums of 24-month-old C57BL/6 mice. Adipocytes have been linked to increased myeloid bias in mice [15Boyd AL Reid JC Salci KR et al.Acute myeloid leukaemia disrupts endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow niche.Nat Cell Biol. 2017; 19: 1336-1347Crossref PubMed Scopus (91) Google Scholar], rhesus monkeys [31Robino JJ Pamir N Rosario S et al.Spatial and biochemical interactions between bone marrow adipose tissue and hematopoietic stem and progenitor cells in rhesus macaques.Bone. 2020; 133115248Crossref PubMed Scopus (3) Google Scholar], and humans [1Aguilar-Navarro AG Meza-León B Gratzinger D et al.Human aging alters the spatial organization between CD34+ hematopoietic cells and adipocytes in bone marrow.Stem Cell Rep. 2020; 15: 317-325Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,15Boyd AL Reid JC Salci KR et al.Acute myeloid leukaemia disrupts endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow niche.Nat Cell Biol. 2017; 19: 1336-1347Crossref PubMed Scopus (91) Google Scholar]. However, the presence of only rare adipocytes in the mouse sternums and in the mouse femoral heads with aging [32Liu LF Shen WJ Ueno M Patel S Kraemer FB Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes.BMC Genom. 2011; 12: 212Crossref PubMed Scopus (82) Google Scholar] may indicate differences in the regulation of steady-state myelopoiesis [33Cuminetti V Arranz L Bone marrow adipocytes: the enigmatic components of the hematopoietic stem cell niche.J Clin Med. 2019; 8: 707Crossref Scopus (20) Google Scholar]. Cellularity assessment was not biased by the difference in hematopoietic cell size across the three species. The cellularity was estimated as the proportion of the nonbone area (composed of hematopoietic parenchyma) to the non-hematopoietic portion (composed of adipocytes). Thus, differences in hematopoietic cell size should not have had a bearing on cellularity. Differences in the number of megakaryocytes between human and mouse bone marrow should be taken into consideration when extrapolating findings from mouse models to humans. Megakaryocytes act as cell-extrinsic factors that seem to influence the behavior of HSCs and their progenitors [34Gorelashvili MG Angay O Hemmen K Klaus V Stegner D Heinze KG Megakaryocyte volume modulates bone marrow niche properties and cell migration dynamics.Haematologica. 2020; 105: 895-904Crossref PubMed Scopus (4) Google Scholar, 35Nakamura-Ishizu A Takubo K Fujioka M Suda T Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin.Biochem Biophys Res Commun. 2014; 454: 353-357Crossref PubMed Scopus (86) Google Scholar, 36Zhao M Perry JM Marshall H et al.Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells.Nat Med. 2014; 20: 1321-1326Crossref PubMed Scopus (326) Google Scholar]. In contrast to a previous study in which an increase in megakaryocyte progenitors was associated with aging in mice [37Rundberg Nilsson A Soneji S Adolfsson S Bryder D Pronk CJ Human and murine hematopoietic stem cell aging is associated with functional impairments and intrinsic megakaryocytic/erythroid bias.PLoS One. 2016; 11e0158369Crossref PubMed Scopus (57) Google Scholar], our study finds that young mouse bone marrow has a higher megakaryocyte content than 24-month-old bone marrow. Our results are in alignment with single-cell RNA sequencing data that indicated that LepR+ cells include different mesenchymal populations that have differential expression of adipogenic, osteogenic, and chondrogenic genes [38Tikhonova AN Dolgalev I Hu H et al.The bone marrow microenvironment at single-cell resolution.Nature. 2019; 569: 222-228Crossref PubMed Scopus (280) Google Scholar]. Hence, a broader distribution of LepR+ BMSCs is expected, including perisinusoidal and periarteriolar [12Ding L Morrison SJ Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches.Nature. 2013; 495: 231-235Crossref PubMed Scopus (750) Google Scholar,39Méndez-Ferrer S Michurina TV Ferraro F et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature. 2010; 466: 829-834Crossref PubMed Scopus (2271) Google Scholar,40Morrison SJ Scadden DT The bone marrow niche for haematopoietic stem cells.Nature. 2014; 505: 327-334Crossref PubMed Scopus (1351) Google Scholar] locations with branching into surrounding parenchyma and around sinusoid-adjacent megakaryocytes. It remains to be elucidated whether the BMSCs that are in different areas of the bone marrow microenvironment correspond to specific subpopulations. The gene expression signatures of human CD45-CD271+ BMSCs are similar to those of specific LepR+ subpopulations in mice [38Tikhonova AN Dolgalev I Hu H et al.The bone marrow microenvironment at single-cell resolution.Nature. 2019; 569: 222-228Crossref PubMed Scopus (280) Google Scholar], suggesting that human CD271+ and mouse LepR+ subpopulations may be equivalent. However, additional studies are needed to address if those populations support lymphoid and myeloid hematopoiesis equally. A feature of our study is that, based on availability, we included different trabecular bones from mice (sternum) than from dogs (femoral neck) and humans (posterior iliac crest). However, these different sources have the common feature of being enriched for trabecular bone. We used mouse sternum because, unlike mouse femur, it is a flat bone that contains trabecular bone fairly similar to that in human bones with active hematopoiesis, including the posterior iliac crest. In situ studies such as those we undertook have some advantages over flow cytometric studies that require cell dissociation. For example, immunomorphology using immunohistochemistry assays enables qualitative and quantitative analyses of protein expression in relation to cell type/morphology, subcellular compartments, and overall distribution of different cell types, including even rare subpopulations. Normal human bone marrow represents a challenge to study because of its low availability. In this study, we used bone marrow from patients undergoing hip replacement. We found no differences among the underlying etiologies or any pre-clinical conditions (data not shown). Osteoarthritis-related bone marrow lesions associated with cystic degeneration or overlying cartilage defects have been reported to be associated with increased CD271+ marrow stromal cell (MSC) density and altered MSC gene expression profile [41Campbell TM Churchman SM Gomez A et al.Mesenchymal stem cell alterations in bone marrow lesions in patients with hip osteoarthritis.Arthritis Rheumatol. 2016; 68: 1648-1659Crossref PubMed Scopus (64) Google Scholar], and indeed, a subset of CD271+ MSCs appear to participate in new bone formation in advanced osteoarthritis [42Ilas DC Baboolal TG Churchman SM et al.The osteogenic commitment of CD271+CD56+ bone marrow stromal cells (BMSCs) in osteoarthritic femoral head bone.Sci Rep. 2020; 10: 11145Crossref PubMed Scopus (6) Google Scholar]. The bone marrow areas that we evaluated were by contrast histologically normal. Nevertheless, it is possible that patients with osteoarthritis may have subtle changes to their bone marrow microenvironment at sites distant from disease involvement. Collectively, our findings indicate that BMSC density and distribution do not vary between human, mouse, and dog and does not change with aging. However, there are significant differences in the microarchitecture of trabecular bone marrow between species, with human bone marrow being significantly more like dog than mouse bone marrow in terms of cellularity and megakaryocyte and adipocyte content (Figure 7). This study also highlights that differences may occur between different mouse strains in key aspects such as megakaryocyte content. In conclusion, CD271+ human and dog and LepR+ mouse BMSCs are highly comparable with respect to density and distribution, supporting the similarity between these mammalian species and the use of the mouse as a suitable in vivo model for HSC–BMSC interactions. However, they might have different functional differences and support lymphoid and myeloid cells with different efficiencies. The differences in adipocyte and megakaryocyte content in the mice need to be taken into consideration and further explored. Overall, our study suggests that dog bone marrow is more similar to human than mouse bone marrow. This work was supported by grants from the Princess Margaret Cancer Centre Foundation (JE Dick), Ontario Institute for Cancer Research with funding from the Province of Ontario (JE Dick), Canadian Institutes for Health Research, Joint Canada Israel Health Research Program of the IDRC (JE Dick and E Flores-Figueroa), Canadian Cancer Society Research Institute (JE Dick), Terry Fox Research Institute PPG (JE Dick), and a Canada Research Chair (JE Dick). This work was carried out with the aid of a grant from the International Development Research Centre (IDRC), Ottawa, Canada. The views expressed herein do not necessarily represent those of the IDRC or its Board of Governors. EM Dorantes-Acosta and A Escobar-Sánchez acknowledge funds from the Hospital Infantil de Mexico (HIM/2013/034/ SSA. 1173). This article constitutes part of the Postgraduate Production Science and Animal Health program, National Autonomous University of Mexico (UNAM). B Meza-León acknowledges the scholarships from the National Council of Science and Technology (CONACyT) and the Mexican Institute of Social Health (IMSS). E Flores-Figueroa received a scholarship from Programa de Cooperación Internacional (IMSS). We thank Ricardo Esquivel, Beremiz Sánchez, Alvaro Zugarazo, Samuel Medina, and Ariel Meza for providing bone marrow samples; Victor Pérez, Jorge Anaya, and Monica Reynoso for technical support; Anastasia Tikhonova for discussion of the article; and Laboratorio Nacional de Microscopía Avanzada-IMSS for the slide scanning service.
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