Bone Marrow Adipocytes

A defining feature of BMA is the storage of lipid within bone marrow adipocytes. We define these cells as a population of bona fide adipocytes, that is, a cell type whose main functional and morphological characteristic is the storage and metabolism of lipids in a single large or several smaller triglyceride-filled vacuoles. This distinguishes adipocytes from other cell types, such as hepatocytes and myofibers, that in principle are also able to store triglycerides ectopically. In these latter cells, lipid accumulation is thought of as predominantly pathological, presumably due to lipotoxic reactions. In addition, haematopoietic stem cells and osteoblasts are also capable of lipid uptake (1–3). However, unlike these other cell types, mature adipocytes are uniquely equipped for metabolising and storing triglycerides and intermediates of lipid metabolism and feature a higher level of resistance to lipotoxicity. We would therefore also recommend against use of less-specific or even colloquial terms, including “fatty cell” or “fat cell” in a combination with the bone marrow, as these may refer to any type of lipid-containing cell (Table 3 and Supplementary Table 2).

A number of abbreviations to specifically define bone marrow-resident adipocytes have been used in the recent literature on BMA. Our recommendation is to maintain consistency with the broader context of adipocyte biology and bone histomorphometry, where adipocytes are commonly abbreviated with “Ad” (5). We therefore propose the consistent use of the terms “BMAd” or “BM adipocyte” to designate mature adipocytes within the bone marrow (Table 3 and Supplementary Table 2).

In addition to their ability to store lipids, another important property of BMAds is secretion of bioactive factors. This is reminiscent of both white and brown adipocytes, which release hormones, lipid species, cytokines, and other factors to exert local and systemic effects (79). Collectively, such adipocyte-derived secreted factors are known as “adipokines” and these contribute extensively to the physiological and pathological functions of adipose tissue (79). BMAds are also becoming increasingly recognised for their ability to secrete adipokines and thereby exert paracrine and endocrine functions (80). The two most prominent adipokines are the hormones leptin and adiponectin, which regulate energy homeostasis and have other diverse effects (79). BMAds express and secrete leptin both in primary culture and after in vitro differentiation from human bone marrow stromal cells (BMSCs) (81–83). BMAds also express and secrete adiponectin and might influence circulating concentrations of this adipokine (80, 84, 85). This suggests that BMAds might have endocrine functions.

BMAds also secrete many other endocrine and paracrine factors, including RANKL (86–88), DPP-4 (89), and stem cell factor (SCF) (90); cytokines such as interleukin-6 (IL-6), IL-3, IL-8, tumor necrosis factor-α (TNF-α), CXCL1, CXCL2, CXCL12, and MCP-1 (91–95); lipid species, such as free fatty acids (96–98); and RNA molecules within extracellular vesicles (80). Through these factors BMAds are reported to modulate haematopoiesis and skeletal remodelling. A full discussion of these functions is beyond the scope of this position paper, but more details are available in several recent reviews (80, 99, 100).

Relating to lipid storage, an unresolved question concerns BM adipocytes' unilocular vs. multilocular nature. These properties are traditionally linked to white and brown adipocyte identity, respectively (79, 101). Some studies suggest that BMAds with a brown adipocyte-like phenotype can occur in the bone marrow cavity (102), and brown adipocyte-like phenotypes can be induced in vitro using cell culture models of BMAds, for example after overexpression of FoxC2 (103) or SIRT1 (103). However, recent data indicate that in vitro cell models do not reliably recapitulate the properties of BMAds in vivo (74). Indeed, microarrays show that UCP1 transcripts are not enriched in whole BM of mice or humans (104, 105), nor is Ucp1 expression greater in BMAds vs. white adipocytes of mice (106). Recent work using lineage tracing and genetic models has also demonstrated that BMAds do not express Ucp1 during development or after adrenergic stimulation in mice (107). Similarly, primary BMAd progenitors have very limited, if any, brown adipogenic potential (89), and BMAds in vivo do not undergo cold-induced glucose uptake (108). However, multilocular BMAds do exist and account for around 5% of all BMAds in the long bones of mice (107). Thus, while it seems that BM adipocytes are distinct from brown adipocytes, it is unlikely that this multilocularity is indeed equated to brown adipocyte-like functions of BMAds. This also pertains to BMAd-intrinsic ability for rapid lipid mobilization in response to adrenergic stimulation or other physiological stimuli known to recruit brown adipocytes for their main function, which is thermogenesis. These remain open issues to be assessed in greater detail. Future authors may therefore opt to add further attributes to the term “bone marrow adipocyte” to better define parameters such as number of vacuoles (locularity), and also to reference anatomical localisation or metabolic characteristics. This relates to the recent discussion on distinct types of BMAds, regulated and constitutive, which will be discussed in the section on Subtypes of Bone Marrow Adipocytes.

Bone Marrow Adipose Tissue

One area of debate is whether BMAds are simply a subpopulation of BM cells that constitute a part of BM as a tissue, or whether, collectively, BMAds act as an integrated adipose tissue. References to “adipose tissue” within the BM can be found as early as 1883 (10), and studies from the 1940s and 1950s also proposed “yellow marrow” or “marrow fat” to be an adipose tissue, based on its similar lipid composition with WAT depots (23, 24). In 1967, Zakaria and Shafrir studied explants of “yellow bone marrow” (YBM), concluding: “The experiments demonstrate the capacity of YBM to synthesize fatty acids and glycerol from glucose, to take up and esterify long-chain FFA (free fatty acids) from an external medium and to release FFA under hormonal stimulation. All these activities are typical of adipose tissue function. Thus, the YBM seems to represent a metabolically active variety of fat store, similar to depots in other anatomical sites, but presumably with a specialized local importance” (31). These studies support the concept that BMAds, collectively, form an integrated adipose tissue.

One caveat, discussed further in the section on Subtypes of Bone Marrow Adipocytes, is that BMAd characteristics vary depending on skeletal site. The studies described above focussed on the yellow marrow, in which BMAds form a contiguous unit that is morphologically similar to white adipose tissue (109). However, BMAds also exist interspersed among the red marrow, where they do not form a spatially contiguous grouping; it is less clear whether these cells, designated “regulated” BMAds, can be considered as an adipose tissue.

This issue was debated extensively among the members of the Nomenclature WG. After much discussion, we concluded that it is appropriate to refer to BMAds as an adipose tissue, even for those adipocytes interspersed among the red marrow. This decision is based on two key points. Firstly, even in white adipose tissue, adipocytes comprise <25% of the total cell population (110). Thus, the fact that BMAds do not predominate in the red marrow does not preclude these from being considered as an adipose tissue. Secondly, “tissue” has been defined as “an aggregation of similarly specialized cells united in the performance of a particular function” (5); hence, possessing a common function is more important than the physical grouping of the cells. Although the roles of BMAds are still being elucidated, it is clear that these cells work together to perform common functions (109), and therefore they can be considered as an integrated adipose tissue.

A second point of debate regards how to abbreviate “bone marrow adipose tissue.” By far the most common abbreviations for this are “MAT” and “BMAT” (Table 4). One benefit of “MAT” is that this is more consistent with other abbreviations in the adipose field, such as “WAT” and “BAT.” One downside to “BMAT” is that this also been used to refer to a “bone marrow aspirate and trephine” biopsy (111); however, there are several benefits to the use of “BMAT” to abbreviate “bone marrow adipose tissue.” Firstly, by using “BM,” “BMAT” refers unambiguously to the BM and is consistent with other abbreviations recommended in this standardised nomenclature (e.g., BMA, BMAd, BMSC, BMFF). Secondly, in the BMA literature the use of “BMAT” precedes use of “MAT” by 5 years, and therefore there is a historical priority for “BMAT” (Table 4). Finally, use of “BMAT” has been recommended in a previous editorial (112), which provides further rationale for its continued use. In summary, we recommend the term “bone marrow adipose tissue” and the abbreviation “BMAT.” This can be defined as a collection of BMAds, which may be clustered together or interspersed among the haematopoietic marrow, that work together to perform common functions.

Two other terms are also recommended. Despite the longstanding references to adipose tissue within the BM, it has historically been more common to refer to “marrow fat” or “bone marrow fat” (Table 4). These terms continue to be used to this day, often in clinical reports and/or to reflect gross measurements of BMA (Supplementary Table 3). Considering the historical prominence and continued use of these terms, we have included “marrow fat” and “bone marrow fat (BM fat)” in the standardised BMA nomenclature. However, when discussing the formation and function of BMAds as a collective, integrated tissue, use of “bone marrow adipose tissue (BMAT)” should be given preference.

Further details of these recommendations, and the many terms that we recommend to avoid using, are provided in Table 4 and Supplementary Table 3.

Subtypes of Bone Marrow Adipocytes

One important concept in BMA research is that BMAds and BMAT display distinct characteristics depending on skeletal location. This was first shown in 1965 by Cohen and Gardiner, who found that during starvation in rabbits, lipid is mobilised from BMAds within the proximal red marrow but not from BMAds in the distal yellow marrow (28). Subsequent work in the 1970s extended these observations, including the demonstration of distinct lipid composition in proximal vs. distal BMAds [reviewed in (113)]. This concept was relatively overlooked until the mid 2010s, when Scheller et al. proposed the existence of “regulated” and “constitutive” subtypes of BMAds (73, 114). Further details, including the evidence supporting these two subtypes, are provided in an excellent recent review (113). Therein, regulated BMAds are “defined histologically as single adipocytes interspersed within the haematopoietic BM. They form gradually throughout life and accumulate with aging.” In contrast, constitutive BMAds “form early in development, are larger in size, and appear histologically as densely packed groups of adipocytes with little intervening haematopoiesis” (113).

The terms regulated and “constitutive” have since gained traction in the field, as evident through the increasing use of the abbreviations “rMAT” or “rBMAT” and “cMAT” or “cBMAT” to refer to these subtypes (Table 5). However, this classification raises an important question: are there only two general classes, or is the heterogeneity less binary than this? For example, in the mouse tibia, proximal (“regulated”), and distal (“constitutive”) BMAds display different properties, in particular in terms of fatty acid saturation, but, like their “regulated” counterparts, the “constitutive” BMAds can also be altered in certain contexts (115, 116). Moreover, in some models of lipodystrophy there is a loss of cBMAT at the distal tibia, whereas cBMAT in caudal vertebrae is maintained (117). Thus, like rBMAT, cBMAT can also display plasticity in response to environmental cues, and cBMAT properties vary depending on skeletal site. Finally, most studies of cBMAT and rBMAT are based in animal models; hence, it remains unclear to what extent these exist as distinct subtypes in humans.

Because of these complexities, as a priority we recommend referring to BMAT and BMAd subtypes based on their anatomical location, in preference to using the “constitutive” and “regulated” terminology. This approach, which already is common in the literature (Table 5), provides a definitive, unambiguous description that addresses the issue of site-specific characteristics. However, given the increasing use of “constitutive” and “regulated,” and the evidence supporting existence of these two broad subtypes (113), we accept that there is still value in continued use of these terms. Therefore, if authors do wish to use these terms, we recommend using rBMAT and cBMAT to refer to the regulated and constitutive subtypes of BMAT, and rBMAd and cBMAd when referring to these subtypes of BM adipocytes.

Further details of these recommendations, and the many terms that we recommend to avoid using, are provided in Table 5 and Supplementary Table 4.

Progenitors for Bone Marrow Adipocytes

BMAds originate from marrow-derived skeletal stem cells (SSCs) (72, 118). These cells go by different names, based on two different concepts regarding their differentiation properties and their tissue of origin. The first concept emanates from in vivo transplantation studies. These studies confirm the existence of multipotent progenitors present in BM stroma that can generate heterotopic bone/marrow organs (ossicles) with donor-derived skeletal cell phenotypes (chondrocytes, osteoblasts, stromal cells), including BMAds [reviewed in (119)]. The term Bone Marrow Stromal Cell (BMSC) is a time-honoured term used to indicate the population of non-hematopoietic, non-endothelial, rapidly adherent cells isolated from BM; adherence in culture is one characteristic property of these stromal cells. Clonal analysis of BMSCs coupled with in vivo transplantation revealed the presence of a subset of multipotent cells (120, 121). Later, these multipotent cells were found to be sinusoidal pericytes, cells that wrap around blood vessels providing them with stability. Furthermore, they were found to restore the perivascular compartment from which they originate (the ability to self-renew), making these cells bona fide SSCs (122). Thus, SSCs are a multipotent, self-renewing subset of BMSCs: all SSCs are BMSCs, but not all BMSCs are SSCs. It is essential to emphasise this because these terms have been used inconsistently in the literature, causing confusion about the relationship between SSCs and BMSCs.

Notably, the term “skeletal stem cell” currently describes a biological activity rather than a well-defined cell phenotype, since the specific identity of multipotent and self-renewing SSCs is not yet clear. As discussed in the accompanying BMAS Methodologies position paper by Tratwal et al., agreement on how to purify SSCs has not yet been reached: even populations highly enriched by using certain cell surface markers are still not homogeneous. Nonetheless, the use of the term “SSC” is appropriate based on the retrospective evidence of stemness demonstrated by the generation of ossicles by clonal populations of BMSCs. However, it is important to note that other populations of SSCs have recently been identified in the growth plate and in the periosteum in both mice and humans, but it does not appear that SSCs from these origins contribute to BMA [reviewed in (123)] (104, 123–126). Similarly, stromal cells capable of adipogenesis ex vivo have been isolated from cortical bone of the femoral diaphysis and cancellous/cortical bone of the proximal epiphysis of rats (127, 128). Although it remains to be confirmed if these cells can generate BMAds in vivo, their ex vivo adipogenic capacity differs depending on skeletal site. This echoes the site-specific differences in BMAd subtypes discussed in the previous section. Thus, the tissue origin of SSCs must always be identified. Indeed, not all populations of SSCs give rise to adipocytes, as determined by in vivo transplantation or lineage tracing in mice. Of note, “in vivo” is the operative term here, as it is known that the in vitro assay for adipogenesis is prone to artefact. In many fibroblastic populations, a few cells accumulate fat from serum in the medium, but do not synthesize triglycerides or hydrolyse them to the same extent as bona fide adipocytes.

According to the second concept, the term “Mesenchymal Stem Cell (MSC)” was introduced, based on the ability of BMSCs/SSCs to make cells and tissues of mesodermal origin. These include not only skeletal cell types (chondrocytes, osteoblasts and BMAds) but also non-skeletal phenotypes such as myoblasts, tenocytes, fibrocytes (ligament), white adipocytes, and others (124). However, as defined by developmental biologists, mesenchyme is an embryonic connective tissue that makes connective tissue, blood, and blood vessels during foetal development, and is not found in post-natal tissue (129). In spite of this, it was proposed that “MSCs” reside in all post-natal tissues, based on non-specific cell surface markers shared by virtually all fibroblastic cells (130), and that all “MSCs” are pericytes (125), able to give rise to osteoblasts, chondrocytes and adipocytes, making them equivalent to SSCs within the BM. However, during embryonic development, none of the non-skeletal cell types, including pericytes, have a common embryonic origin (118). Lastly, the “MSC” term emerged from less-than-rigorous in vitro studies; more-recent in vivo work has confirmed that the progenitor activity of different populations of perivascular stromal cells (if any) is always restricted to that of the tissue of origin (126).

For these reasons, the use of the term “Mesenchymal Stem Cell” and any other definition including the word “Mesenchymal,” especially the use of the acronym “BMSC” to indicate bone marrow mesenchymal stem cells rather than bone marrow stromal cells, is not scientifically accurate and is not recommended. Thus, we recommend using “SSCs” to refer to “Skeletal Stem Cells” and “BMSCs” to refer to “Bone Marrow Stromal Cells.” Describing the anatomical location from which these cells are isolated, as well as the species of origin, is also important when reporting studies of SSCs and BMSCs.

Further details of these recommendations, and the terms and abbreviations that we recommend to avoid using, are provided in Table 6 and Supplementary Table 5.

BMAT Morphometric Analyses

The elaboration of an appropriate nomenclature for BMAT morphometry cannot be conceived without considering the guidelines for bone histomorphometry that have been established for more than 25 years (5, 131). Indeed, many of the terms usable for BMAT morphometry are well-defined and properly abbreviated in the first Standardized Nomenclature, Symbols, and Units for Bone Histomorphometry (131) and in its revision (5).

As for bone, BMAT morphometry can be based on two- or three-dimensions (2D or 3D) and applied to many types of biological material, most commonly iliac crest bone/BM biopsies obtained from human subjects, and bones from experimental animals. Recent studies have also analysed BMAds within BM plugs of transgenic mice, allowing study of BMAd development by lineage tracing (132). A mixture of terminology for 2D and 3D techniques of analysis should not be used in the same article, unless different methods of analysis (i.e., histology vs. micro-CT) are used (5).

Both primary measurements and referents (i.e., some clearly defined area or volume within the sample) must be considered. The former include Adipocyte Area (Ad.Ar), Volume (Ad.V), Perimeter (Ad.Pm), Diameter (Ad.Dm), and Number (N.Ad); each of these abbreviations is consistent with the guidelines for bone histomorphometry (5). In addition to adipocyte number, measurements of the size of individual adipocytes (Ad.Ar, Ad.V, Ad.Pm, Ad.Dm) are important in the physiology and pathology of BMAT. Indeed, changes in total BMAT may be the result of mechanisms that lead to the increase or decrease of either BMAd size (lipid storage/lipolytic activity) or number (adipogenic differentiation, or BMAd apoptosis and clearance). Size-related measurements may have meaning per se and not require a referent. For example, they can be used for the calculation of mean (and median) of BMAds and to construct distribution curves; the latter provide a more accurate representation of BMAd sizes across the sample and therefore are the recommended method of presenting such data (Table 7).

In contrast to these measurements of BMAd size, for N.Ad, total adipose tissue area and total adipose volume, referents are needed, and must be properly defined. Since BM is distributed throughout the cavities of the skeleton and includes distinct kinds of tissues/cells (haematopoietic cells, BMAds, and BMSCs) and is highly vascularised, the referents may be different. Some of them (i.e., Tissue Area in 2D and Tissue Volume in 3D) have been already defined and abbreviated (T.Ar and TV, respectively) in the Standardized Nomenclature, Symbols, and Units for Bone Histomorphometry (5). For studies of BMA, additional referents to be considered include Marrow Area (Ma.Ar, in 2D) and Marrow Volume (Ma.V, in 3D), which coincide with the spaces of the skeleton delimited by endosteal surfaces (cancellous bone surfaces and endocortical surfaces).

The choice between T.Ar (or TV) or Ma.Ar (or Ma.V) as referents is important for the accurate interpretation of the data. For example, when osteopenia does occur, the Ma.Ar (or Ma.V) increases while T.Ar (or TV) does not. In addition, since haematopoietic cells and adipocytes are located in the Extra-Vascular compartment of the BM, the use of this region (Area: ExVa.Ma.Ar, i.e., Marrow Area minus Vascular Area; Volume: ExVa.Ma.V, i.e., Marrow Volume minus Vascular Volume) as referent could be more appropriate and, in principle, to be preferred in respect to Ma.Ar and Ma.V. Indeed, this Extra-Vascular compartment can be used as a referent not only for assessments of BMA (i.e., with total Ad.Ar, total Ad.V, or N.Ad as the numerator), but also for measuring the percentage of haematopoietic marrow. The latter is calculated by expressing the haematopoietic area (Hm.Ar) or haematopoietic volume (Hm.V) relative to ExVa.Ma.Ar or ExVa.Ma.V, respectively. As discussed in the accompanying BMAS Methodologies paper, the ratio of haematopoietic to adipose area (Hm.Ar./Ad.Ar.) may also be of interest in some instances but should not be confounded with measures of adiposity (e.g., Ad.Ar/Ma.Ar) or hematopoietic cellularity (e.g., Hm.Ar/Ma.Ar).

One challenge is that measurement of the Extra-Vascular Marrow compartment can be highly time-consuming. Thus, we suggest to use the area (or volume) of the Extra-Vascular Marrow compartment as referent in specific conditions only, for example in mouse long bone whole-mount sections in which the vascular spaces (sinusoids and central vein) may constitute a very significant and dynamic area or volume (133); and, in humans, in those haematological diseases in which BM vascularity is known to be altered (134). Use of the Extra-Vascular Marrow referent is, however, still limited by technological challenges, which may resolve as whole-mount 3D microscopy and high-resolution contrast-enhanced μCT become more widely available to reconstruct, and subtract, the vascular network within the BM space (135, 136). Subtraction of the central vein from the Marrow Area is, however, common in whole-bone histology of murine long bones due to the dilated lumen often associated with retraction artefacts upon fixation (reviewed in the accompanying BMAS Methodologies position paper by Tratwal et al.).

A final consideration, debated extensively among WG members, is whether to use the term “Adipocyte density” or “Adipocyte number” when describing measurements of N.Ad relative to a referent region (i.e., Ma.Ar, Ma.V, T.Ar, TV, ExVa.Ma.Ar, or ExVa.Ma.V). Such measurements report the population density of BMAds within the region of interest, and therefore we recommend that these are reported as “Adipocyte density” (Table 7). However, one concern with the term ‘density' is that readers may confuse this with a measurement of the actual physical density of BMAds (i.e., mass per unit volume of the cells); this concern was raised in the published guidelines for bone histomorphometry (5), which therefore prioritises the term “number” over “density” when reporting relative cell numbers. Thus, to be consistent with these previous guidelines, we agree that use of “Adipocyte number” is also acceptable when describing measurements of N.Ad relative to a referent region. What is essential is that studies should never report measurements of N.Ad alone: a suitable referent must always be used.

Details of the proposed nomenclature regarding BMAT morphometry are presented in Table 7, with further information in Supplementary Table 6.

MRI- and/or MRS-Based Analyses

MRI analysis of BMA was first reported in the mid-1980s (53–57), but the past 20 years have seen a notable increase in publications using MRI and/or 1H-MRS for assessment of BMA (Table 8 and Supplementary Table 7). Most studies report the fat fraction (FF) or lipid:water ratio, calculated from the lipid and water peaks in MR spectra (71); however, there is much inconsistency in how these measurements are reported. As shown in Table 8, measurements described by the term “fat fraction (FF),” or the related terms “marrow fat fraction” or “bone marrow fat fraction (BMFF),” typically are reported as a %, but in some cases the actual measurement reported is the lipid/water ratio and not the % fat fraction. Conversely, sometimes measurements described as a “ratio” are actually reported as % fat fraction. Confusing issues further, some studies have used the term “fat content” when reporting measurements of % fat fraction, whilst others have used this term when reporting the lipid/water ratio. In other cases, the terms “fat fraction” or “fat content” are used, but the units of measurement are not stated; this may cause confusion about whether the measurement is a fraction or a ratio. Finally, MRI has been used to report the volume or area of BMA from 3D or 2D images, respectively (Table 8).

This variability underscores the need for standardisation. To date, most studies use the term “fat fraction,” “marrow fat fraction,” or “bone marrow fat fraction” to report the % fat fraction of the BM (Table 8). These measurements typically are based on dual-echo MRI or MRS data, which can be subject to confounding factors (e.g., attenuation of the T2^* signal) (71). In contrast, measurement of the proton density fat fraction (PDFF), based on multi-echo data, removes the effect of these confounding factors and thereby represents a truly standardised BMA measurement (71). Thus, in addition to 1H-MRS, PDFF is emerging as another gold-standard for BMA quantification. Nevertheless, many studies report dual-echo fat fraction measurements. We recommend reporting MRI and 1H-MRS-based measurements as fat fraction (FF) or bone marrow fat fraction (BMFF) when based on dual-echo data, and as PDFF when based on appropriate multi-echo sequences. These measurements should be reported as %, rather than the lipid/water ratio.

A final issue is the ability of MR to measure the degree of lipid saturation. Terms such as “unsaturation index” (UI) and “unsaturated lipid fraction” (ULF) have been used (137–141), and this capacity to assess not just BMAT quantity, but also BMAT quality, may reveal further insights about the clinical implications of altered BMA (71, 99). However, compared to reports of FF, BMFF or PDFF, there are far fewer papers reporting BMAT saturation, and therefore we feel it is too early to recommend terms, abbreviations or units for describing such measurements.

Further details of these recommendations, and the terms that we recommend to avoid using, are provided in Table 8 and Supplementary Table 7.

A special thank you to Olaia Naveiras, Ecole Polytechnique Fédérale de Lausanne, for help on aligning this manuscript with the accompanying BMAS methodologies position paper, and to all members of the BMAS Scientific Board for providing feedback on these nomenclature recommendations. Finally, in our systematic review of the BMA literature we have strived to include all relevant publications; however, given the scope of this effort, we may have missed some papers relevant to this field. We apologise to any authors whose work we have inadvertently overlooked.