In human, an association between bone loss and increased marrow adipose tissue suggests that medullary adipocytes could play a role in osteoporosis by acting on neighboring bone-forming osteoblasts. Supporting this hypothesis, we previously showed, using coculture and conditioned medium models based on human bone marrow stromal cells of commercial origin, that factors secreted by adipocytes induced the transdifferentiation of osteoblasts towards an adipocyte-like phenotype. The aim of this study was to confirm the involvement of medullary adipocyte secretion products in the alteration of osteoblasts in pathophysiological conditions. To this end, two new cellular models were developed from human bone biopsies representing the aging skeleton. In the first model, outgrowth from trabecular bone fragments were used to isolate primary cells that displayed a specific osteoblast phenotype. We confirmed the transdifferentiation of these primary osteoblasts following their incubation with adipocyte conditioned medium as evidenced by the increase in the levels of adipogenic mRNA markers (PPARG, Leptin and HSD11B1, P < 0,001) and the decrease of osteogenic transcript (BGLAP, P < 0.05). The second model was based on primary adipocyte isolation through collagenase treatment followed by ceiling and 2D culture. Oil red O staining confirmed the isolation of fully-differentiated primary adipocytes from bone biopsies. Experiments performed with their conditioned medium validated what had been observed with primary osteoblasts: the effect of the adipocyte secretome on osteoblast fate. Overall, our results supported the relevance of these models to examine paracrine interactions between osteoblasts and adipocytes and confirmed the role played by adipocyte-secreted factors in osteoblast transdifferentiation.

Sclerostin is a Wnt signaling pathway inhibitor that negatively regulates bone formation. Bone-marrow-derived stromal cell (BMSC) differentiation is influenced by the Wnt pathway, leading to the hypothesis that higher levels of sclerostin might be associated with an increase in bone marrow adiposity (BMA). The main purpose of this study was to determine whether a relationship exists between circulating sclerostin and BMA in post-menopausal women with and without fragility fractures. The relationships between circulating sclerostin and body composition parameters were then examined. The outcomes measures included vertebral and hip proton density fat fraction (PDFF) using the water fat imaging (WFI) MRI method; DXA scans; and laboratory measurements, including serum sclerostin. In 199 participants, no significant correlations were found between serum sclerostin and PDFF. In both groups, serum sclerostin was correlated positively with bone mineral density (R = 0.27 to 0.56) and negatively with renal function (R = -0.22 to -0.29). Serum sclerostin correlated negatively with visceral adiposity in both groups (R = -0.24 to -0.32). Serum sclerostin correlated negatively with total body fat (R = -0.47) and appendicular lean mass (R = -0.26) in the fracture group, but not in the controls. No evidence of a relationship between serum sclerostin and BMA was found. However, serum sclerostin was negatively correlated with body composition components, such as visceral adiposity, total body fat and appendicular lean mass.

In human, bone loss is associated with increased marrow adipose tissue and recent data suggest that medullary adipocytes could play a role in osteoporosis by acting on neighboring bone-forming osteoblasts. Supporting this hypothesis, we previously showed, in a coculture model based on human bone marrow stromal cells, that factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this work, we employed an original integrative bioinformatics approach connecting proteomic and transcriptomic data from adipocytes and osteoblasts, respectively, to investigate the mechanisms underlying their crosstalk. Our analysis identified a total of 271 predicted physical interactions between adipocyte-secreted proteins and osteoblast membrane protein coding genes and proposed three pathways for their potential contribution to osteoblast transdifferentiation, the PI3K-AKT, the JAK2-STAT3 and the SMAD pathways. Our findings demonstrated the effectiveness of our integrative omics strategy to decipher cell-cell communication events.

Background Systemic inflammation is the main factor underlying secondary osteoporosis in patients with rheumatoid arthritis (RA). The JAK inhibitors, such as Tofacitinib (Tofa), can control systemic inflammation and have beneficial effects on bone in various models. This might be due to direct effects on the bone microenvironment and not exclusively based on their anti-inflammatory function. Bone marrow adipocytes (BMAds) are abundant in the bone microenvironment. The effect of JAK inhibitors on BMAds is unknown, but evidence suggests that there is competition between human bone marrow-derived stromal cell (hBMSCs) differentiation routes toward BMAds and osteoblasts (Ob) in osteoporosis. Objectives To determine in various models whether Tofa influences directly bone marrow cell committment toward adipogenesis and osteoblastogenesis. Then, in a prospective pilot study, to investigate the potential effects of Tofa on bone marrow adiposity in patients with RA. Methods To study the effect of Tofa on cellular commitment, hBMSCs were cultured for 3 days in appropriate Ob- and BMAd- differentiation media (Ob-3d and BMAd-3d), together with Tofa at 200, 400 (equivalent to a therapeutic dose of 5 mg twice a day in RA patients) or 800 nM. To mimic inflammatory conditions, TNFα was added to the media at a dose of 1 ng/ml. This study was also conducted on mature BMAds and a similar treatment was applied for 6 days to mature BMAds at 14 days of differentiation (BMAd-20d). The impact of Tofa was determined by gene expression profile analysis, western-blot analysis and cell density monitoring. In parallel, in a pilot study of 9 RA patients treated with Tofa 5 mg twice a day (TOFAT study: NCT04175886 ), proton density fat fraction (PDFF) was measured by MRI (Dixon technique) at the lumbar spine at the start of treatment and at 6 months. Results In non-inflammatory conditions, the gene expression of Runx2 decreased in Ob-3d treated with Tofa 400 and 800 nM(p<0.05). Conversely, BMAd-3d treated with Tofa (at 200, 400 and 800 nM) exhibited a substantial increase in the gene expression of PPARγ2, C/EBPα and Perilipin 1 (a marker associated with lipid droplet formation) compared to controls (p<0.05). The increase in the expression of Perilipin 1 was also confirmed at the protein level. In inflammatory conditions, BMAd-3d and Ob-3d markers decreased considerably (PPARy2 and RUNX2, respectively, p<0.05), but the addition of Tofa did not change the expression profiles of Ob-3d compared to TNFα controls. On the contrary, the analysis of PPARy2 gene expression showed that Tofa limited the negative effect of TNFα on BMAd differentiation (p<0.05). The positive effect of Tofa on mature adipocyte (BMAd-20d) under inflammatory conditions was also supported by an increase in the density of differentiated BMAds (p<0.001). These findings were consolidated by an increase in PDFF at 6 months of treatment with Tofa in RA patients (+6.9%, p<0.01). Conclusion Overall, in vitro and clinical results suggest a stimulatory effect of Tofa on BMAds commitment and differentiation, which does not support a positive effect of Tofa on bone Disclosure of Interests Jean-Guillaume Letarouilly Consultant of: Sêmeia, Grant/research support from: Pfizer, Julien Paccou: None declared, Sammy Badr: None declared, René-Marc Flipo Consultant of: member of the advisory board Pfizer, Christophe Chauveau: None declared, Bernard Cortet: None declared, Odile Broux: None declared, Aline Clabaut: None declared

BackgroundSystemic inflammation is the main factor underlying secondary osteoporosis in patients with rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi), such as tofacitinib (Tofa), can control systemic inflammation and may have beneficial effects on bone in various models. This might be due to direct effects on the bone microenvironment and not exclusively based on their anti-inflammatory function. Bone marrow adipocytes (BMAds) are abundant in the bone microenvironment. The effect of JAKi on BMAds is unknown, but evidence suggests that there is competition between human bone marrow-derived stromal cell (hBMSC) differentiation routes towards BMAds and osteoblasts (Ob) in osteoporosis.ObjectivesThe aims of the study are to determine whether Tofa influences BMAds and Ob derived from hBMSCs and to investigate the potential effects of Tofa on bone marrow adiposity in RA patients.MethodsTo determine the effect of Tofa on cellular commitment, hBMSCs were differentiated to BMAds or OBs for 3 days together with Tofa at 200, 400, or 800 nM and TNFα. This study was also conducted using differentiated BMAds. The impact of Tofa was determined by gene and protein expression analysis and cell density monitoring. In parallel, in a pilot study of 9 RA patients treated with Tofa 5 mg twice a day (NCT04175886), the proton density fat fraction (PDFF) was measured using MRI at the lumbar spine at baseline and at 6 months.ResultsIn non-inflammatory conditions, the gene expression of Runx2 and Dlx5 decreased in Ob treated with Tofa (p <0.05). The gene expression of PPARγ2, C/EBPα, and Perilipin 1 were increased compared to controls (p <0.05) in BMAds treated with Tofa. Under inflammatory conditions, Tofa did not change the expression profiles of Ob compared to TNFα controls. In contrast, Tofa limited the negative effect of TNFα on BMAd differentiation (p <0.05). An increase in the density of differentiated BMAds treated with Tofa under TNFα was noted (p <0.001). These findings were consolidated by an increase in PDFF at 6 months of treatment with Tofa in RA patients (46.3 ± 7.0% versus 53.2 ± 9.2% p <0.01).ConclusionTogether, these results suggest a stimulatory effect of Tofa on BMAd commitment and differentiation, which does not support a positive effect of Tofa on bone.

L’inflammation chronique dans la polyarthrite rhumatoïde (PR) est un déterminant majeur de la survenue d’une ostéoporose secondaire. Le tofacitinib (Tofa), un inhibiteur de la voie de signalisation JAK-STAT, est capable de contrôler l’activité inflammatoire chez les patients atteints de PR et son mécanisme d’action pourrait conduire à des interactions directes avec l’os [1], [2]. L’objectif principal de ce travail était de déterminer l’impact du Tofa sur les cellules du microenvironnement osseux dans des conditions inflammatoires. En particulier, nous avons étudié son effet sur la différenciation des cellules stromales dérivées de la moelle osseuse humaine (hBMSCs) vers les cellules ostéoblastiques (Ob) mais aussi vers les adipocytes médullaires (Ad) car le déséquilibre compétitif entre l’adipogenèse et l’ostéoblastogenèse est un processus mécanistique clé dans l’ostéoporose. Les hBMSCs (RoosterBio) ont été différenciées en Ob ou Ad dans des milieux appropriés pendant 3 jours avec le Tofa, utilisé à 200 nM, 400 nM (équivalent de la posologie de 5 mg ×2/jour) et 800 nM, et avec 1 ng/mL de TNFα afin de reproduire la condition d’inflammation dans la PR. Pour évaluer l’effet du Tofa sur des Ad matures, un traitement similaire a été appliqué pendant 6 jours sur des Ad dérivés des hBMSCs à 14 jours de différenciation. Les effets du Tofa ont été déterminés par un test de viabilité, une analyse d’expression génique et des tests biochimiques. En parallèle, dans une étude pilote, l’adiposité médullaire via la fraction graisseuse a été mesurée par IRM (technique Dixon) initialement et à 6 mois chez 9 patients atteints de PR traités par tofacitinib (étude TOFAT : NCT04175886). Les tests de cytotoxicité ont démontré que la viabilité cellulaire était très peu affectée sous Tofa et/ou TNFα. En condition non-inflammatoire et après 3 jours de différenciation Ob, le Tofa utilisé à 400 nM a conduit à une diminution significative de 2 fois l’expression génique de Runx2 (facteur de transcription clé de l’ostéogenèse) (p < 0,05). Au contraire, après trois jours de différenciation, les Ad traités sous Tofa 200, 400 et 800 nM, présentaient une forte augmentation de l’expression génique de PPARγ2, C/EBPα (deux facteurs de transcription clés de la différenciation des Ad) et de la Perilipine 1 (marqueur associé à la formation de gouttelettes lipidiques) par rapport au contrôle (p < 0,05). En condition inflammatoire, les niveaux d’expression génique de Runx2 n’étaient pas affectés quel que soit le traitement Tofa appliqué. Cependant, le traitement Tofa semblait limiter l’action négative du TNFα sur la différenciation des Ad. En effet, le niveau d’expression génique de PPARγ2 a été fortement augmenté avec la combinaison de TNFα et de Tofa 200, 400 et 800 nM par rapport aux Ad traitées par TNFα seul (p < 0,05). Cet effet positif sur l’adipogenèse en condition inflammatoire était aussi soutenu par une augmentation de la densité des Ad différenciées après traitement sous Tofa. Par ailleurs, nous avons retrouvé une augmentation significative de la fraction graisseuse à six mois de traitement par tofacitinib chez 9 patients atteints de PR (46,2 ± 7,0 % contre 53,2 ± 9,2 %, p < 0,01). Les données suggèrent une action stimulatrice du Tofa sur l’engagement et la différenciation des Ad. Ces résultats in vitro sont corrélés par des données cliniques préliminaires. L’augmentation de l’adiposité médullaire observée sous Tofa ne semble pas en faveur d’un effet positif du traitement sur l’os.

Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment.The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblastmediated bone formation.The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts.Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism.It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging.Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis.In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.

A shift in the commitment of human skeletal stem cells (SSCs) from the osteogenic lineage to the adipogenic lineage can result in increased marrow adiposity and bone loss. Advances in understanding the fate decision of SSCs and particularly the intracellular mechanisms controlling bone marrow adipocyte (BMA) differentiation have thus relevance to bone disorders. The aim of this review is to report the recent contributions of Omics studies to the understanding of mechanisms controlling human BMA differentiation.

The development of a bone metastasis involves interactions between the tumor cells, the bone marrow microenvironment and the bone cells themselves. A better understanding of the pathophysiological changes occurring in bone metastasis can be obtained from histopathological examination of invaded specimens. This review focuses on the main molecular mechanisms implied in the localization and growth of malignant cells in the bone marrow. The corresponding histologic developmental stages are illustrated both in osteolytic (or mixed metastasis) or in the osteosclerotic forms by histological analysis, immunohistochemistry and microcomputed tomographic analysis of bone samples. In both cases, the malignant cells find a “fertile soil” in the bone marrow microenvironment. They use the growth factors released by bone cells for the coupling between osteoclasts/osteoblasts to promote their own development. In turn, they elaborate a variety of cytokines that can promote osteoclastogenesis (PTHrP, IL-1, IL-6…) or on the contrary, other growth factors that can boost the osteoblastic activity (ET1, IGFs). A “vicious circle” occurs between the malignant cells and the bone cells leading to the radiological expression of the metastasis.Le développement d’une métastase osseuse implique des interactions entre les cellules malignes, le microenvironnement médullaire et les cellules osseuses elles-mêmes. L’analyse histopathologique d’échantillons osseux envahis par une métastase permet d’aborder les mécanismes physiopathologiques de la progression tumorale. La présente revue fait le point des principaux mécanismes moléculaires impliqués dans la localisation et le développement des cellules malignes dans l’os. Les stades de progression sont illustrés dans les métastases ostéolytiques (ou mixtes) et dans les formes ostésclérotiques par des clichés microscopiques en coloration standard et immunohistochimie ainsi que par microtomographie aux rayons X. Dans les deux cas, les cellules tumorales trouvent dans le microenvironnement médullaire un « sol fertile » qui va favoriser leur croissance. Elles utilisent les facteurs de croissance libérés par les cellules osseuses (et servant physiologiquement au couplage ostéoclastes/ostéoblastes) comme promoteur de leur propre expansion. En retour, elles libèrent de nombreuses cytokines qui stimulent localement l’ostéoclastogenèse (PTHrP, IL-1, IL-6…) ou, à l’inverse, d’autres facteurs qui augmentent considérablement l’activité ostéoblastique (ET-1, IGFs). Un « cercle vicieux », s’établi entre cellules malignes et cellules osseuses ; il aboutit, in fine, à l’expression radiologique de la métastase.

BACKGROUND:In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model.RESULTS:We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts.CONCLUSIONS:Here, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.

Bone marrow adipocytes (BM Ads) are active and specific adipocytes which modulate marrow microenvironment. BM Ads interact with osteoblasts and osteoclasts and are highly suspected to be involved in primary and secondary osteoporosis. BM Ads also interfere with hematopoiesis and could play a role in several malignant diseases.

In osteoporosis, bone loss is accompanied by greater adiposity in the marrow. Given the cellular proximity within the bone marrow, we wondered whether adipocytes might have a paracrine impact on osteoblast differentiation. To test this hypothesis, we cocultured adipocytes with osteoblasts derived from mesenchymal stem cells (MSCs) in the absence of direct cell contact and then analyzed gene expression changes in the osteoblastic population by using real-time reverse transcription polymerase chain reaction. We found that, upon coculture, MSC-derived osteoblasts showed appearance of adipogenic (lipoprotein lipase, leptin) and decrease of osteogenic (osteocalcin) mRNA markers. Our results indicate that in vitro, MSC-derived adipocytes are capable of inducing MSC-derived osteoblasts to differentiate to an adipocyte phenotype. These new data suggest that (i) transdifferentiation of committed osteoblasts into adipocytes may contribute to the increase in marrow fat content at the expense of bone-forming cells and (ii) this switch might be initiated by the adipocytes themselves.

L'embryogenèse somatique (ES) est une voie asexuée aboutissant à la formation d'embryons à partir de cellules somatiques qui ressemble à l'embryogenèse zygotique. Chez la chicorée, une variabilité génétique pour ia capacité à former des embryons somatiques in vitro a été mise en évidence et un génotype K59. embryogène et K28. peu embryogène ont été sélectionnés pour obtenir une descendance F1' Cette variabilité a été exploitée afin d'identifier les régions chromosomiques ou QTL et les gènes Impliqués dans l'ES. Après 7 jours d'induction des explants racinaires en condition d'embryogenèse et 30 jours de développement, les plantules issues (PL) du développement des embryons somatiques et les structures chlorophylliennes unipolaires (SH) ont été comptabilisées. Les caractères PL et SH présentent une distribution normale et continue et une héritabilité supérieure à 63%. Une carte génétique issue du croisement K28*K59 a été construite pour la recherche de QTL liée à J'ES. Six QTL ont été identifiés. expliquant plus de 23% et 44% de variation phénotypique totale pour les caractères PL et SH respectivement. Parmi les 63 gènes candidats cartographiés, 16 co-localisent avec les QTL pour PL et SH. La co-localisation de gènes homologues à SHOOT MERISTEMLESS (STM) et ARGONAUTE (AGO) d'Arabidopsis avec les QTL6 et QTL2, respectivement. est particulièrement intéressante. En effet. ces gènes sont connus pour leur implication dans la maintenance de l'état dédifférencié des cellules souches dans le méristème caulinaire. Avec la détection des QTL pour l'ES. les résultats de cette étude ont fourni pour la première fois des éléments sur le contrôle génétique de l'ES chez la chicorée.