Bone marrow adipogenic lineage precursors are the major regulator of bone resorption in adult mice

Lu, Jiawei; He, Qi; Wang, Huan; Yao, Lutian; Duffy, Michael; Guo, Hanli; Braun, Corben; Zhou, Yilu; Liang, Qiushi; Lin, Yuewei; Bandyopadhyay, Shovik; Tan, Kai; Choi, Yongwen; Liu, X. Sherry; Qin, Ling

doi:10.1038/s41413-025-00405-4

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Published: 19 March 2025

Bone marrow adipogenic lineage precursors are the major regulator of bone resorption in adult mice

Jiawei Lu^1,2,
Qi He¹,
Huan Wang¹,
Lutian Yao¹,
Michael Duffy¹,
Hanli Guo¹,
Corben Braun¹,
Yilu Zhou¹,
Qiushi Liang¹,
Yuewei Lin¹,
Shovik Bandyopadhyay^3,4,
Kai Tan^3,4,
Yongwen Choi ORCID: orcid.org/0000-0002-1007-7998⁵,
X. Sherry Liu¹ &
…
Ling Qin ORCID: orcid.org/0000-0002-2582-0078¹

Bone Research volume 13, Article number: 39 (2025) Cite this article

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Abstract

Bone resorption by osteoclasts is a critical step in bone remodeling, a process important for maintaining bone homeostasis and repairing injured bone. We previously identified a bone marrow mesenchymal subpopulation, marrow adipogenic lineage precursors (MALPs), and showed that its production of RANKL stimulates bone resorption in young mice using Adipoq-Cre. To exclude developmental defects and to investigate the role of MALPs-derived RANKL in adult bone, we generated inducible reporter mice (Adipoq-CreER Tomato) and RANKL deficient mice (Adipoq-CreER RANKLflox/flox, iCKO). Single cell-RNA sequencing data analysis and lineage tracing revealed that Adipoq⁺ cells contain not only MALPs but also some mesenchymal progenitors capable of osteogenic differentiation. In situ hybridization showed that RANKL mRNA is only detected in MALPs, but not in osteogenic cells. RANKL deficiency in MALPs induced at 3 months of age rapidly increased trabecular bone mass in long bones as well as vertebrae due to diminished bone resorption but had no effect on the cortical bone. Ovariectomy (OVX) induced trabecular bone loss at both sites. RANKL depletion either before OVX or at 6 weeks post OVX protected and restored trabecular bone mass. Furthermore, bone healing after drill-hole injury was delayed in iCKO mice. Together, our findings demonstrate that MALPs play a dominant role in controlling trabecular bone resorption and that RANKL from MALPs is essential for trabecular bone turnover in adult bone homeostasis, postmenopausal bone loss, and injury repair.

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Introduction

Bone is critical for protecting internal organs, supporting the body, allowing movement, as well as hosting hematopoiesis. To maintain its essential structure and functions, bone undergoes continuous remodeling—a cyclic process involving osteoclastic bone resorption and osteoblastic/osteocytic bone formation.¹ In healthy adults, this remodeling process is precisely balanced to preserve normal bone mass. In aged and diseased populations, this balance is shifted towards resorption rather than formation, leading to osteoporosis characterized by low bone mass, deteriorated bone structure, and high risk of fracture.¹ Following injuries, bone remodeling is a crucial process that facilitates the bridging of the fracture gap.²

One important aspect of bone remodeling is to initiate osteoclast formation at the trabecular or cortical bone surface for bone resorption. Descended from myeloid progenitors of the hematopoietic lineage, osteoclasts are highly specific, large multinucleated, and phagocytic cells secreting acid and catalytic enzymes to demineralize and degrade collagen rich bone extracellular matrix (ECM).³ For a long time, they were thought to be short-lived cells that rapidly undergo apoptosis following their fusion from mononuclear progenitors.⁴ Recent in vivo research using advanced intravital imaging techniques discovered that they are actually long-lived cells constantly undergoing recycling through fission and fusion mechanism.^5,6,7 Instead of apoptosis, the fission products, osteomorphs, can refuse among each other or with existing osteoclasts to extend the longevity of osteoclasts.⁶

Past research has pointed out two cytokines as the most important regulatory factors for osteoclast formation and function: colony-stimulating factor (Csf1) and receptor activator of nuclear factor kappa Β ligand (RANKL). The former one promotes the proliferation of osteoclast precursors and their expression of receptor activator of nuclear factor kappa Β (RANK), a RANKL receptor.⁸ The latter one is the predominant factor that drives the differentiation of osteoclast precursors into mature osteoclasts.⁹ It also stimulates osteoclasts to migrate and undergo cycles of fusion and fission in vivo.⁶ Encoded by Tnfsf11, RANKL belongs to tumor necrosis factor (TNF) superfamily and exists in two forms: membrane-bound and soluble.¹⁰ Upon binding to RANK, it initiates the transcription of a cascade of osteoclast specific genes via up-regulating the expression of a master transcription factor nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1).¹¹ Early research identified osteogenic cells, particularly bone matrix-embedded osteocytes, are the major source of RANKL that regulates osteoclast formation.^12,13,14 This finding fits well with the concept of bone remodeling as it emphasizes the crosstalk between bone forming and resorbing cells.

In the past several years, the application of advanced single cell transcriptomics approaches to bone research greatly expanded our knowledge about cellular components of bone tissue and their transcriptome profiles. In particular, single cell RNA-sequencing (scRNA-seq) of mesenchymal lineage cells revealed a unique mesenchymal subpopulation that highly expresses adipogenic markers, including Pparg, Cebpa, Lpl, Adipoq, etc., but does not contain lipid droplets.^15,16 Since they are precursors for mature adipocytes, we termed them marrow adipogenic lineage precursors (MALP).^15,17 Other groups also identified similar cell types and named them Adipo-Cxcl12-abundant-reticular (Adipo-CAR) cells or marrow Adipoq⁺ cells (MACs).^16,18 Interestingly, scRNA-seq suggested that MALPs, but not osteoblasts nor osteocytes, are the major source of RANKL and Csf1 in bone.^19,20 Subsequent studies from our group and other groups confirmed that specific deletion of either factor in adipogenic lineage cells results in a drastically elevated trabecular bone mass due to diminished osteoclasts.^19,20,21,22

However, those studies have limitations because they used a constitutive Cre, Adipoq-Cre, to examine the action of MALPs-derived factors. MALPs emerge in mouse bone right after birth.²³ While those studies analyzed mice up to 6 months of age, it is possible that changes in adult bone is due to developmental defect. In addition, since Adipoq-Cre also labels bone forming cells in adult mice, Adipoq⁺ cells were regarded as bipotent bone marrow skeletal stem/progenitor cells.²⁴ Hence, we cannot exclude the possibility that osteoclast regulatory factors are also depleted in osteoblasts and osteocytes in those studies. To circumvent these limitations, in this study, we first performed a lineage tracing experiment using inducible Adipoq-CreER Tomato (AdipoqER/Td) mice to delineate the relationship between MALPs and Adipoq⁺ cells. Next, we adopted RNA fluorescence in situ hybridization (FISH) to identify RANKL-expressing cells in vivo. Finally, we constructed inducible RANKL deficient mice using Adipoq-CreER and examined their adult bones under normal, estrogen depletion, and injury repair conditions. Our data revealed MALPs as the main source of RANKL in adult mice.

Results

Adipoq⁺ cells contain not only MALPs but also some bipotent mesenchymal progenitors

We previously used Adipoq-Cre to study MALPs in vivo. To examine whether Adipoq⁺ cells contain other progenitors, we integrated scRNA-seq datasets of bone marrow mesenchymal cells from 1- and 16-month-old mice we previously reported (Fig. 1a).¹⁵ Cell clusters were annotated by their marker gene expression (Fig. S1A). Pseudotime trajectory analysis revealed that early mesenchymal progenitors (EMPs) give rise to late mesenchymal progenitors (LMPs) and lineage committed progenitors (LCPs), which are then differentiated into either adipogenic lineage (MALPs) or osteogenic lineage (osteoblasts and osteocytes) (Fig. 1b). Violin plots clearly showed that while Adipoq is highly expressed in MALPs, it is also expressed in LCPs followed by osteoblasts and osteocytes in 1 month dataset at very low levels (Fig. 1c). Interestingly, EMPs express Adipoq and other MALP markers at 16 months of age, albeit their expression levels were much lower than those in MALPs (Fig. 1c, Fig. S1B).

To analyze Adipoq⁺ cells in adult mice, we generated inducible Td reporter mice driven by Adipoq-CreER. These mice, AdipoqER/Td, at 3 months of age received daily Tamoxifen (Tam) injections from day 1 to 3. At day 7, many Td⁺ cells were observed inside the long bone (Fig. 1d(i)). Within the metaphysis region, Td⁺ cells were made of 73.5% ± 1.0% Cd45- stromal cells, 2.7% ± 0.2% Perilipin⁺ adipocytes, 9.0% ± 0.4% pericytes, and 14.8% ± 1.2% bone lining cells [Fig. 1d(ii–v), n = 5 mice]. Although some bone surface lining cells were also Td ⁺ , they did not express Osterix, an osteogenic cell marker [Fig. 1d(v, vi)]. Particularly at the endocortical bone surface, we observed a lot of Td⁺ cells in the close proximity to Osterix⁺ osteoblasts. Td did not label chondrocytes, osteocytes or periosteal cells [Fig. 1d(vi, vii)]. In addition, in situ staining of Pparg, the master transcriptional factor for adipogenic differentiation²⁵ and another marker for MALPs,¹⁵ showed that it is only expressed in Td⁺ cells (Fig. 1e). These data indicate that Adipoq-CreER targets MALPs, but not bone forming cells (osteoblasts and osteocytes). Furthermore, CFU-F assay showed that almost all CFU-F colonies are Td- (Fig. 1f, g), suggesting that Adipoq⁺ cells lack the proliferation ability required by early progenitors.

To determine the fate of Adipoq⁺ cells, we harvested long bones of AdipoqER/Td mice at 1, 4, 8 and 12 weeks post the first Tam injection for lineage tracing experiment (Fig. 2a, b). Perilipin staining revealed that Td labels nearly all mature adipocytes throughout the tracing period. On the contrary, Td gradually labeled osteoblasts and osteocytes over time. While no Td⁺ osteoblasts and osteocytes were detected at 1 week in both trabecular and cortical bone, the percentages of Td⁺ osteoblasts increased to 46.1%, 77.1% and 92.1% and the percentages of Td⁺ osteocytes increased to 9.9%, 12.4% and 27.6% in the trabecular bone at 4, 8 and 12 weeks, respectively. In the cortical bone, almost all endosteal osteoblasts became Td⁺ after 4 weeks but few osteocytes (3.4%) became Td⁺ even after 12 weeks of tracing. These data suggest that Adipoq⁺ cells contain not only committed adipo-lineage cells but also uncommitted mesenchymal progenitors capable of osteogenic differentiation.

We noticed that Td⁺ cells are not evenly distributed through the bone marrow. Thus, we counted them at four anatomic sites: subchondral bone, top metaphysis (region close to the growth plate), bottom metaphysis (region distal to the growth plate), and diaphysis (Fig. 2c). Interestingly, we found that the density of bone marrow Td⁺ cells (excluding bone surface and embedded cells) is much lower in the midshaft region compared to the trabecular bone region. During the 3-month tracing period, Td⁺ cells in the area with high trabecular bone volume (subchondral bone and top metaphysis) remained unaltered, but Td⁺ cells in the area with low trabecular bone volume (bottom metaphysis and diaphysis) decreased significantly (Fig. 2c, d). These data indicate that mesenchymal progenitors labeled by Adipoq-CreER are not early-stage progenitors with self-renewal ability. Together with CFU-F result, our lineage tracing data indicate that Adipoq⁺ cells likely include some late, bipotent mesenchymal progenitors, possibly LCPs identified in scRNA-seq.

MALPs are the major producer of osteoclast regulator factors in adult bone

Our previous scRNA-seq of mouse bone marrow predicted that MALPs are the major producers of osteoclast regulatory factors, including RANKL and Csf1.¹⁵ We recently profiled bone marrow from human femoral heads.²⁶ Cell clustering revealed 6 mesenchymal cell clusters: Fibro-MSC (mesenchymal stromal cell), APOD ⁺ MSC, Adipo⁻MSC, THY1 ⁺ MSC, Osteo⁻MSC and Osteoblast (Fig. 3a). Among these clusters, Adipo⁻MSC and THY1 ⁺ MSC highly expressed adipogenic genes, and a major difference between them is THY1 expression. Thus, we consider them both as human counterpart of MALPs (Fig. 3b). In line with mouse data (Fig. S2), RANKL (TNFSF11) was mainly expressed in THY1⁺ MSCs, while CSF1 was mainly expressed in Adipo⁻ and THY1⁺ MSCs followed by Fibro⁻MSCs. Their expression was much lower in osteolineage cells than in MALPs. We also examined their expression in other bone marrow cells (Fig. S3). While RANKL expression was restricted to mesenchymal lineage cells, CSF1 expression was broader, including megakaryocyte-erythroid progenitors (MEPs), erythroblasts, basophil/eosinophil/mast (Ba/Eo/Ma) cells, blood vessel cells etc. However, the highest expression was still detected in Adipo-MSCs.

To confirm this finding, we stained Rankl in situ on 3-month-old AdipoqER/Td mouse femurs harvested at day 7 after the first Tam injection. Interestingly, almost all Rankl-expressing cells were Td⁺ (Fig. 3c, n = 5 mice). Most of them resided in the metaphyseal and diaphyseal bone marrow and some were on the trabecular and cortical bone surface. Moreover, co-staining showed that Rankl⁺ cells were also Pparg⁺ cells (Fig. 3d). On the contrary, only 60.2% ± 0.8% of Csf1-expressing cells were Td⁺ (Fig. 3e, n = 5 mice). Importantly, we did not observe any Rankl and Csf1 mRNA expression in osteocytes in either trabecular bone or cortical bone (Fig. 3c, e). Similarly, in human bone, RANKL expression was mainly detected in PPARG-expressing cells in the bone marrow (Fig. S4, n = 3), demonstrating that MALPs, but not osteogenic cells, are the major cell source of osteoclast regulatory factors.

MALP-derived RANKL supports bone resorption in adult mice

To investigate the role of MALP-derived RANKL in adult bone remodeling, we constructed Adipoq-CreER RANKL^flox/flox (RANKL iCKO) mice. At 3 months of age, these mice displayed similar trabecular and cortical bone structures in femurs and vertebrae as WT siblings (Fig. S5). Next, we subjected both WT and iCKO mice to Tam injections for 3 days. Four weeks later, Rankl mRNA were reduced by 70.0% and 2 weeks later, RANKL protein amount were reduced by 71.6%, respectively, in bone marrow from iCKO mice but not in the cortical bone (Fig. 4a, b). This change did not alter their body weight (Fig. S6A) and longitudinal bone growth, as indicated by growth plate thickness and femoral bone length (Fig. S6B–D). Strikingly, compared to WT mice, iCKO mice exhibited a 3.3-fold increase in trabecular bone volume fraction (BV/TV), a 1.8-fold increase in trabecular number (Tb.N), a 2.0-fold increase in trabecular thickness, and a 69.7% decrease in trabecular separation (Tb.Sp) at 4 weeks post Tam injections (Fig. 4c–e). These mice gained trabecular bone mass quickly as the increase of BV/TV can be detected as early as 2 weeks post Tam injections (Fig. S7A, B). On the contrary, the cortical bone structure of iCKO mice was not altered (Fig. S8). Similar massive bone gain phenotype was also observed in vertebrae (Fig. S9).

We next performed bone histomorphometry to uncover the cellular changes. TRAP staining revealed that osteoclasts are greatly reduced by 44.7% and 63.0% at the trabecular bone surface at 2 and 4 weeks post Tam injections, respectively, but not changed at the chondro-osseous junction (COJ) between growth plate and primary spongiosa and endosteal bone surface (Fig. 4f, g, Fig. S7C, D). Meanwhile, osteoblasts (Osterix⁺ bone surface cells) was decreased by 12.5% and 17.4%, respectively (Fig. 4h, i, Fig. S7E, F) and their activity was also significantly reduced (Fig. 4j, k). Serum chemistry confirmed those changes, showing a 34.7% reduction in bone resorption marker CTX-1 and a 14.2% reduction in bone formation marker P1NP (Fig. 4l). Overall, these data show that MALP-derived RANKL is important for maintaining bone resorption in adult mice.

RANKL not only regulates bone metabolism but also hematopoietic cells.⁹ Since RANKL is expressed in MALPs that are distributed throughout the bone marrow, we examined hematopoietic cells in iCKO mice. However, flow analysis did not detect any changes in hematopoietic components in the bone marrow or peripheral blood (Fig. S10A, B). Their spleen weight was not altered either (Fig. S10C), suggesting that hematopoiesis is normal in iCKO mice.

RANKL depletion in MALPs attenuates ovariectomy (OVX)-induced bone loss

OVX surgery in mice mimics human postmenopausal osteoporosis. To understand the functional role of MALP-derived RANKL in pathological bone loss, we injected Tam into 3-month-old female WT and iCKO mice for 3 days and subjected them to sham or OVX surgery one day after the last injection. Mice were euthanized 6 weeks later. Estrogen deficiency was confirmed by an 86.7% decrease in uterine weight of WT (Fig. S11). Similar changes were also observed in iCKO mice. In sham groups, iCKO mice displayed a drastically increase in femoral and vertebral trabecular bone mass (2.9-fold and 1.5-fold, respectively) compared to WT mice (Fig. 5a, b, Fig. S12). OVX reduced femoral trabecular BV/TV by 57.8% in WT mice and 36.9% in iCKO mice. Compared to WT mice, iCKO mice exhibited 4.5-, 1.9- and 1.8-fold increases in BV/TV, Tb.N, and Tb.Th, respectively, and a 70.3% decrease in Tb.Sp at 6 weeks post OVX (Fig. 5a, b). This preservation of trabecular bone post OVX was more prominent in vertebrae, with 53.4% and 25.8% decreases in BV/TV in WT and iCKO mice (Fig. S12), respectively. OVX did not affect femoral cortical bones in both WT and iCKO mice (Fig. S13).

Bone histomorphometry revealed that OVX increased osteoclast surface in both WT and iCKO mice but iCKO mice with OVX have 49.8% less osteoclast surface compared to WT mice with OVX (Fig. 5c, d). OVX also increased osteoblast surface and osteoblast activity in WT and iCKO mice (Fig. 5e–g). Serum chemistry further confirmed that bone turnover is increased in both genotypes but bone resorption, marked by CTX-1, is 37.2% less in iCKO with OVX compared to WT with OVX (Fig. 5h). Taken together, the above data demonstrate that RANKL from MALPs contributes to the enhanced bone resorption in the OVX model.

OVX also induces bone marrow adiposity (Fig. 5i, j). Interestingly, while MALPs are precursors for marrow adipocytes, their number did not change after OVX (Fig. S14). Compared to WT, we did not observe any change in marrow adipocytes in iCKO mice after sham surgery. After OVX, adipocyte area and size in iCKO mice were increased similarly as WT mice (Fig. 5i, j). These data suggest that RANKL from MALPs does not participate in OVX-induced marrow adiposity.

RANKL depletion in osteoporotic bone restores bone mass

Next, we investigated whether MALPs-derived RANKL can be targeted for osteoporosis treatment. To do so, we subjected 3-month-old mice to OVX. Six weeks later when trabecular bone mass is significantly reduced, iCKO mice received vehicle or Tam injections for 3 days to deplete RANKL expression in MALPs. As a control, WT mice received similar surgery and injections. To our surprise, even 3 times of Tam injections significantly increased femoral and vertebral trabecular bone mass in WT mice by 1.6-fold and 1.5-fold, respectively, at 4 weeks later (Fig. 6a, b, Fig. S15), suggesting that Tam alone has beneficial effects on bone. In comparison, Tam administration increased femoral trabecular bone mass in iCKO mice at a much higher level (3.3-fold), accompanied by a 1.5-fold increase in Tb.N, a 2.0-fold increase in Tb.Th. and a 49.0% decrease in Tb. Sp (Fig. 6a, b). Similar effects were also observed in vertebral trabecular bone (Fig. S15).

Subsequent bone histomorphometry revealed that Tam injections in WT mice decrease osteoclast surface by 18.7% (Fig. 6c, d) while increasing osteoblast surface by 1.1-fold and enhancing osteoblast activity (Fig. 6e–g). Strikingly, Tam injections in iCKO mice greatly reduced osteoclast surfaces by 53.1% (Fig. 6c, d). Osteoblast surface was reduced by 9.6% (Fig. 6e, f) and osteoblast activity was also reduced (Fig. 6g). Serum chemistry confirmed that iCKO mice have a greater reduction of bone resorption than WT mice after Tam treatment (Fig. 6h). Taken together, our data suggest that after OVX-induced osteoporosis is established, depletion of RANKL in MALPs is still effective in restoring trabecular bone within a short period of time.

MALP-derived RANKL contributes to bone healing after injury

Osteoclasts play an important role in the cartilage and bone remodeling stages of fracture healing.² However, whether they are also required for healing after bone defect injury is not well studied. Since Adipoq⁺ cells are located inside the bone, not at the periosteal bone surface, we next drilled non-critical size holes in the femoral cortex of iCKO and WT mice. In this injury model, trabecular bone appears first in the bone marrow close to the cortical defect region and then is resolved after healing, indicating a bone remodeling process. Meanwhile, the defect area is filled with new bone via intramembranous ossification. We carried out the drill-hole injury on mice 4 days after daily Tam injections at day 1-3. MicroCT analysis showed that the hole in WT mice is healed nicely at 4 weeks post injury with almost no intramedular trabecular bone left (Fig. 7a, b). However, iCKO mice still had a significant amount of trabecular bone remaining. Compared to WT mice, iCKO mice showed decreased BV/TV in the cortical bone area (25.7%) and increased BV/TV in the intramedullary area (2.0-fold), indicating a delayed healing. Histomorphometry analysis showed that osteoclasts in iCKO mice are drastically reduced by 62.7% in the defect cortical bone area and 78.0% in the intramedular trabecular bone area (Fig. 7c, d), while osteoblasts are not affected (Fig. 7e, f). Our data indicate that MALP-derived RANKL drives osteoclastogenesis and bone remodeling in this type of bone repair.

Discussion

Our prior studies, as well as others, revealed that MALPs-derived osteoclast regulatory cytokines, RANKL and Csf1, are important for trabecular bone remodeling in young mice.^19,20,21,22 However, those studies used constitutive Adipoq-Cre and thus did not address their actions in adult bone tissue. In this report, we first analyzed mouse and human scRNA-seq datasets and utilized in situ experiments to discover that RANKL and Csf1 are mainly expressed in MALPs but not in osteoblasts and osteocytes. We then studied adult bone phenotypes of RANKL deficient mice at two anatomic sites (long bone and vertebra) using inducible Adipoq-CreER under normal and pathological conditions. Collectively, our data demonstrate that RANKL derived from marrow adipogenic precursors plays a dominant role in stimulating osteoclast formation and promoting trabecular bone resorption under normal and pathological conditions. This conclusion is consistent with a previous report revealing that adipogenic transcription factors C/EBPβ and δ promotes RANKL expression in mesenchymal progenitors undergoing adipogenic differentiation.²⁷

Prior studies using osteocyte-specific Cres, such as Dmp1-Cre and Sost-Cre, to ablate RANKL proposed that bone embedding osteocytes are crucial for trabecular bone remodeling.^12,13,14 Our research challenges this conventional view. First, scRNA-seq of bone marrow mesenchymal lineage cells in mouse and human samples revealed a specific expression of RANKL in MALPs. Second, in situ staining of Rankl clearly showed that Rankl is mainly expressed in cells expressing Adipoq and Pparg, two markers for MALPs. To our surprise, we did not detect Rankl mRNA in osteocytes, which might reflect a relatively low sensitivity of in situ approach. A prior report detected RANKL expression in one third of osteocytes using RANKL antibody.²⁸ However, their immunohistology images showed more RANKL⁺ cells in the bone marrow. Third, RANKL iCKO mice exhibited a striking 3.3-fold increase of femoral trabecular bone mass within one month of RANKL depletion, which is higher than ~2.5-fold increase in 6-month-old Dmp1-Cre RANKL CKO mice.¹³ At this time point, only 9.9% of osteocytes and 46.1% of osteoblasts in the trabecular bone are labeled by Td in AdipoqER/Td mice. Fourth, Dmp1-Cre is not specific for osteocytes. Lineage tracing revealed that it also labels all osteoblasts and ~30% CAR cells,²⁹ a mesenchymal subpopulation highly overlapped with MALPs. These data are in line with the low Dmp1 expression in LCP and osteoblast clusters in our scRNA-seq.¹⁷ Thus, it is likely that Dmp1-Cre driven RANKL knockout depletes RANKL in MALPs as well. Sost-Cre is more specific for osteocytes. However, it also labels many hematopoietic cells.¹² Some of them, such as B and T lymphocytes, express RANKL too.^30,31,32 Lastly, our proposal that MALPs are a predominant source of RANKL in the trabecular bone also fits well with the emerging view that osteoclasts are long-lived cells constantly undergoing recycling.³ Using intravital microscope, McDonald et al. discovered that RANKL rapidly stimulates fusion and fission of osteomorphs, small daughter cells of osteoclasts.⁶ Since osteomorphs reside only in the bone marrow, MALPs in the bone marrow but not osteocytes in the bone matrix are more likely to be the major RANKL source regulating their activities. That being said, we cannot exclude the importance of osteocytes-derived RANKL in diseases and treatment conditions not investigated in this research. Due to their abundance in the cortical bone, osteocytes are likely to be the major regulator of cortical bone turnover. This is supported by findings from Xiong et al., which demonstrated that mice with Dmp1-Cre-driven RANKL knockout are resistant to tail suspension–induced cortical bone loss.¹³

In addition to osteoporosis, osteoclasts are also important for bone healing. Past research in this field focused on fracture, which is mostly repaired via an endochondral ossification mechanism.³³ Previous studies showed that suppressed bone resorption, either by RANK depletion³⁴ or by pharmacological inhibition of RANKL,³⁵ delays cartilage dissolution and callus remodeling and thus reduces bony unions. On the contrary, increased RANKL activity by depleting OPG, the decoy receptor for RANK, stimulates osteoclastogenesis and accelerates bone fracture healing.³⁶ To our knowledge, this study is the first investigation of osteoclasts in bone healing after drill hole injury, which is repaired via an intramembranous ossification mechanism. It is interesting to note that RANKL depletion in MALPs does not affect cortical bone during bone maintenance and after estrogen deficiency but delays cortical bone healing after drill hole injury. In RANKL iCKO mice, reduced osteoclastogenesis at the injury site causes persistent remaining of bony callus, leading to delayed healing at the injury site.

Bone surface is covered by osteoblasts, osteoclasts, and bone lining cells. Compared to osteoblasts with a large, cuboidal shape, bone lining cells are morphologically defined as flattened cells covering quiescent bone surface not undergoing bone remodeling. In the conventional view, they are descendants of osteoblasts and able to be quickly re-activated into osteoblasts upon stimulations, such as PTH, mechanical loading, and radiation.^37,38,39 A prior study also found that they can be a major source of osteoblasts during adulthood.⁴⁰ However, it is puzzling that scRNA-seq analyses performed so far have not identified a subpopulation matching the above characteristics of bone lining cells. To our surprise, we found many Td⁺ cells on the trabecular and endocortical bone surface in adult AdipoqER/Td mice. Those Adipoq⁺ bone surface cells are Osterix- cells, and hence not osteoblasts. Some of them express Pparg, Rankl, or Csf1 mRNAs, which are highly specific for MALPs based on scRNA-seq. These data suggest that bone lining cells may contain adipogenic cells. Future research using spatial omics techniques will define the composition of bone lining cells and provide new insights into bone remodeling.

One limitation of our study is that Adipoq-CreER does not solely label MALPs. In our study, Adipoq-CreER instantly marks adipocytes and Pparg-expressing cells while gradually marking osteoblasts and osteocytes over time. This indicate that in addition to MALPs, it also labels mesenchymal progenitors capable of bilineage differentiation. The same labeling pattern has been reported by other researchers.^23,24 Since Adipoq⁺ cells do not form CFU-Fs and do not expand over time, those additional bipotent progenitors are likely to be late-stage mesenchymal progenitors, such as LCPs identified in our scRNA-seq. This leads to a possible depletion of RANKL in osteogenic cells in our mouse model. Nevertheless, since our studies focus on 2-6 weeks after Tam injection, a time point when the majority of osteocytes are not labeled by Td, we believe our conclusion that MALP-derived RANKL plays a dominant role in trabecular bone resorption is still valid.

In conclusion, we have demonstrated that bone marrow adipoprogenitors control bone resorption at the trabecular bone region in adult mice during homeostasis and pathological conditions. Currently, Denosumab, an FDA-approved human monoclonal antibody to RANKL, is the first line osteoporosis drug.⁴¹ It also shows great promise in other diseases, such as bone tumors and autoimmune diseases. Our research discovering that MALPs are the major source of RANKL in adult bone marrow sheds new insight into targeting this important signaling pathway in various diseases. With the advance in drug design and delivery, future studies should focus on how to manipulate this cell population to achieve the best clinical efficacy with minimum side effects.

Material and methods

Analysis of scRNA-seq datasets

Pre-aligned scRNA-seq matrix files were acquired from GEO GSE145477 and GSE176171 (mouse) and GSE253355 (human). Standard Seurat pipeline⁴² was used for filtering, normalization, variable gene selection, dimensionality reduction analysis and clustering. For the integrated dataset, batch integration was performed using Harmony (version 1.0).⁴³ Cell type was annotated according to the metadata from published datasets.^15,26

Animals study design

All animal work performed in this report was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. Adipoq-CreER Rosa-tdTomato (AdipoqER/Td) mice were generated by breeding Rosa-tdTomato⁴⁴ mice with Adipoq-CreER mice.⁴⁵ To generate RANKL iCKO mice, we first bred Adipoq-CreER with RANKL^flox/flox mice²⁰ to obtain Adipoq-CreER RANKL^flox/+, which were then crossed with RANKL^flox/flox to generate RANKL iCKO mice. Male RANKL iCKO mice was further crossed with female RANKL^flox/flox mice to generate RANKL iCKO mice and WT (RANKL^flox/flox) siblings. All mouse lines, except RANKL^flox/flox, were obtained from Jackson Laboratory (Bar Harbor, ME, USA). To induce Td expression and RANKL depletion, mice at 3 months of age received daily intraperitoneal injections of Tam (75 mg/kg) for 3 days. For OVX surgery, 3-month-old female mice received either OVX or sham operation and their femurs, tibiae, and vertebrae were collected 6 or 10 weeks later for analyses. For drill hole injury, 3-month-old female mice received a 0.8-mm diameter unicortical drill hole defect via a 21 G needle at the diaphysis part of right femurs and their injured femurs were collected 4 weeks later for analyses.

Micro-computed tomography (microCT) analysis

MicroCT analysis (microCT 45, Scanco Medical AG, Brüttisellen, Switzerland) was performed at 7.4 µm isotropic voxel size as described previously.⁴⁶ Briefly, the distal end of femur corresponding to a region at 0–3.4 mm below the growth plate was scanned. The images of the secondary spongiosa regions (0.6–2.1 mm below the lowest point of the growth plate (GP), ~200 slices) were contoured for trabecular bone analysis. At the femur midshaft, 100 slices located at 4.7–5.5 mm away from the distal growth plate were acquired for cortical bone analyses. In vertebrae, the region 50 slices away from the top and bottom end plates (~300 slices) was acquired for trabecular bone analysis. To analyze bone healing after drilling a hole, the contouring of defect area or intramedullary area were manually defined. A total of 150 slices were used for trabecular bone analysis. Trabecular and cortical bones were segmented from soft tissue using a threshold of 487.0 mgHA/cm³ and 661.6 mgHA/cm³, respectively, with a Gaussian noise filter (sigma = 1.2, support = 2.0). For trabecular bone analysis, trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were recorded. For cortical bone analysis, periosteal perimeter (Ps.Pm), endosteal perimeter (Ec.Pm), cortical bone area (Ct.Ar), cortical thickness (Ct.Th), and tissue mineral density (TMD) were recorded. All calculations were performed based on 3D standard microstructural analysis.⁴⁷

Human sample collection

Femoral heads were de-identified surgical discard specimens obtained from 3 patients undergoing total hip arthroplasty surgery. Morphologically normal bone marrow tissues were sampled at the distal site to the articular cartilage for histology analysis.

Histology

To obtain cryosections without decalcification, mouse bones were dissected and fixed in 4% paraformaldehyde (PFA) for 24 h, dehydrated in 30% sucrose, embedded in optimal cutting temperature (OCT) compound, and sectioned at 6 μm in thickness using cryofilm tape (Section Lab, Hiroshima, Japan). For immunostaining, sections were incubated with rabbit anti-Osterix (Abcam, ab22552), rat anti-CD45 (Biolegend, 103101), rat anti-Endomucin (Santa Cruz, sc-65495) or rabbit anti-Perilipin (Cell signaling, 9349) at 4 °C overnight followed by Alexa Fluor 488 anti-rat (Abcam, ab150155) or Alexa Fluor 647 anti-rabbit (Abcam, ab150075) secondary antibodies incubation 1 h at RT. Fluorescent TRAP staining was performed as described previously.⁴⁸ Sections were scanned by Axioscan (Carl Zeiss MicroImaging, Göttingen, Germany). In the lineage tracing experiment, we selected the following areas in distal femurs to count Td⁺ bone marrow cells: subchondral bone, top metaphysis (0.6–2.1 mm distal to GP), bottom metaphysis (3.1–4.6 mm distal to GP), and diaphysis (6.5–8.0 mm distal to GP).

To measure dynamic histomorphometry, mice received calcein (10 mg/kg, Sigma Aldrich) and xylenol orange (90 mg/kg, Sigma Aldrich) at 9 and 2 days, respectively, before euthanization. Areas within the secondary spongiosa of tibiae were quantified by OsteoMeasure Software (OsterMetrics, Decatur, GA, USA). The primary indices include total tissue area (TV), trabecular bone perimeter (BS), single- and double-labeled surface (s/dLS), and interlabel width. Mineralizing surface (MS), bone formation rate (BFR), and surface-referent bone formation rate (BFR/BS, μm³/μm²/d) were calculated as described by Dempster et al.⁴⁹

To obtain paraffin sections, femurs were fixed in 4% PFA for 24 h and decalcified in a 10% EDTA for 4 weeks at 4 °C. Samples were then embedded in paraffin, sectioned at 6 μm in thickness, and processed for H&E staining and Safranin O/fast green staining.

After collection, human bone samples were fixed in 4% PFA overnight, decalcified by Morse solution (10% sodium citrate and 22.5% formic acid) for 3 days, after rinsing the samples three times with RNase-Free water, proceed with paraffin embedding for sectioning.

For RNA FISH experiment, we adopted in situ hybridization chain reaction (HCR) approach (Molecular Instruments, Los Angeles, CA). For mouse samples, cryosections were processed and stained by probes against Rankl (NM_011613.4), Csf1 (NM_001113529.1), and Pparg (NM_001127330.3) mRNAs according to manufacturer’s protocol (HCR™ RNA-FISH protocol for fresh frozen or fixed frozen tissue sections). For human samples, paraffin sections were stained by Human probes against RANKL (NM_003701.4), PPARG (NM_001330615.4) mRNAs.

Hematopoietic phenotyping

Bone marrow was flushed from mouse femurs and pre-treated with Fc-blocker (Invitrogen, 14-0161-81). After washing, bone marrow cells were stained with CD45 AF700 (Biolegend, 103205), CD170 FITC (Biolegend, 155503), Ly6G APC (Biolegend, 127605), CD115 PE-CY7 (Biolegend, 135523), Ly6C Percp (Biolegend, 128027), and CD11b BV605 (Biolegend, 563015). Peripheral blood cells were collected from mouse tail vein, processed for red blood cell lysis using PharmLyse (BD Pharmingen, 555899). To analyze T cells and B cells, peripheral blood cells were stained with CD45 AF700 (Biolegend, 103205), CD11b BV605 (Biolegend, 563015), CD3 FITC (Biolegend, 100203) and B220 Percp (Biolegend, 103233). To analyze myeloid lineage, cells were stained with CD45 AF700 (Biolegend, 103205), CD170 FITC (Biolegend, 155503), Ly6G APC (Biolegend, 127605), CD115 PE-CY7 (Biolegend, 135523), Ly6C Percp (Biolegend, 128027), and CD11b BV605 (Biolegend, 563015). Flow cytometry experiments were performed by BD LSRFortessa flow cytometer and analyzed by FlowJo v10.5.3 for WIN.

Colony-forming unit fibroblast (CFU-F) assay

Bone marrow cells were flushed from mouse long bones and seeded at 3 × 10⁶ cells per T25 flask in growth medium (α-MEM supplemented with 15% FBS, 0.1% β-mercaptoethanol, 20 mmol/L glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin) for 7 days before counting CFU-F number under the fluorescence inverted microscope (Leica, Germany) using bright field and fluorescence channel.

ELISA assays

Sera were collected during mouse euthanization for measuring bone turnover markers, collagen type I C-telopeptide degradation products (mouse CTX-I ELISA Kit, MyBioSource), N-terminal propeptide of type I procollagen (Immunotag™ Mouse PINP ELISA Kit, G-Bioscience) according to the manufacturer’s instructions. Bone marrow collected by centrifugation was used to measure RANKL amount (PeproTech, 900-K233K).

qRT-PCR analysis

Bone marrow was centrifuged from long bones and mixed with Tri Reagent (Sigma Aldrich) for RNA purification. Cortical bone was dissected from the remaining marrow-free bones, crushed in liquid nitrogen, mixed and homogenized with Tri Reagent on ice (Sigma Aldrich) for RNA purification. A Taqman Reverse Transcription Kit (Applied BioSystems, Inc., Foster City, CA, USA) was used to reverse transcribe mRNA into cDNA. The power SYBR Green PCR Master Mix Kit (Applied BioSystems, Inc) was used for quantitative real-time PCR (qRT-PCR). Primers for Tnfsf11 gene are 5’- GGAAGCGTACCTACAGACTA-3’ (forward) and 5’- TGCTCCCTCCTTTCATCA-3’ (reverse), and primers for β-actin gene are 5’- TCCTCCTGAGCGCAAGTACTCT-3’(forward) and 5’-CGGACTCATCGTACTCCTGCTT-3’ (reverse).

Statistical analyses

Data are expressed as means ± standard deviation (SD). For comparisons between two groups, unpaired two-sample Student’s t test was applied. For comparisons amongst multiple groups across two fixed effect factors (e.g., genotype and surgery), two-way ANOVA was applied, followed by Tukey-Kramer multiple comparison test to account for family-wise type I error using Prism 8 software (GraphPad Software). Values of P < 0.05 were considered statistically significant.

Data availability

All the data support the figures and the other findings are available upon reasonable request to the corresponding author.

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Acknowledgements

We thank MicroCT Imaging Core at Penn Center for Musculoskeletal Disorders (PCMD) for their assistance with microCT analysis. We also thank Dr. Jesse Williams at University of Minnesota for his assistance with bone marrow and peripheral blood analysis. This study was supported by NIH grants NIH/NIA R01AG069401 (to L.Q.), NIH/NHLBI U54HL165442 (to K.T.), and P30AR069619 (to Penn Center for Musculoskeletal Disorders).

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Authors and Affiliations

Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
Jiawei Lu, Qi He, Huan Wang, Lutian Yao, Michael Duffy, Hanli Guo, Corben Braun, Yilu Zhou, Qiushi Liang, Yuewei Lin, X. Sherry Liu & Ling Qin
Department of Spine Surgery, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200092, China
Jiawei Lu
Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
Shovik Bandyopadhyay & Kai Tan
Center for Childhood Cancer Research, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
Shovik Bandyopadhyay & Kai Tan
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
Yongwen Choi

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Jiawei Lu
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Qi He
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Huan Wang
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Lutian Yao
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Qiushi Liang
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Yuewei Lin
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Shovik Bandyopadhyay
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Kai Tan
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Contributions

Jiawei Lu conducted the experiments and drafted the manuscript. Qi He, Huan Wang, Corben Braun, and Yuewei Lin performed the experiments. Lutian Yao, Michael Duffy, Yilu Zhou, Qiushi Liang, and Shovik Bandyopadhyay contributed to data analysis. Hanli Guo was responsible for animal husbandry. Kai Tai, Yongwen Choi, and Sherry Liu conceptualized and designed the study and supervised the research. Ling Qin oversaw the project, contributed to study design, supervised the research, secured funding and played a key role in drafting and revising the manuscript.

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Correspondence to Ling Qin.

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Lu, J., He, Q., Wang, H. et al. Bone marrow adipogenic lineage precursors are the major regulator of bone resorption in adult mice. Bone Res 13, 39 (2025). https://doi.org/10.1038/s41413-025-00405-4

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Received: 26 July 2024
Revised: 28 December 2024
Accepted: 20 January 2025
Published: 19 March 2025
DOI: https://doi.org/10.1038/s41413-025-00405-4