Abstracts BMAS Summer School 2023-2nd Bone Marrow Adiposity Society Summer School Meeting 2023.

Fat is the main component of an adult bone marrow and constitutes the so-called bone marrow adipose tissue (BMAT). Marrow adipocytes, which are the fat cells in the bone marrow, become more abundant with age, and may influence the whole-body metabolism. In osteoporotic patients, the amount of BMAT has an inverse correlation with the amount of bone mass. In people with anorexia nervosa that lose weight after the reduction of peripheral adipose tissues, BMAT expands. Although bone marrow adipocytes are increasingly recognized as a target for therapy, there is still much to learn about their role in skeletal homeostasis, metabolism, cancer, and regenerative treatments. The Bone Marrow Adiposity Society (BMAS), established in 2017, aims to enhance the understanding of how BMAT relates to bone health, cancer, and systemic metabolism. BMAS is committed to training young scientists and organized the second edition of the BMAS Summer School, held on September 4-6, 2023, as a virtual event.

Young minds, deeper insights: a recap of the BMAS Summer School 2023, ranging from basic research to clinical implications of bone marrow adipose tissue.

Bone marrow adiposity (BMA) is a rapidly growing yet very young research field that is receiving worldwide attention based on its intimate relationship with skeletal and metabolic diseases, as well as hematology and cancer. Moreover, increasing numbers of young scientists and students are currently and actively working on BMA within their research projects. These developments led to the foundation of the International Bone Marrow Adiposity Society (BMAS), with the goal to promote BMA knowledge worldwide, and to train new generations of researchers interested in studying this field. Among the many initiatives supported by BMAS, there is the BMAS Summer School, inaugurated in 2021 and now at its second edition. The aim of the BMAS Summer School 2023 was to educate and train students by disseminating the latest advancement on BMA. Moreover, Summer School 2023 provided suggestions on how to write grants, deal with negative results in science, and start a laboratory, along with illustrations of alternative paths to academia. The event was animated by constructive and interactive discussions between early-career researchers and more senior scientists. In this report, we highlight key moments and lessons learned from the event.

The Impact of Interventional Weight Loss on Bone Marrow Adipose Tissue in People Living with Obesity and Its Connection to Bone Metabolism.

This review focuses on providing physicians with insights into the complex relationship between bone marrow adipose tissue (BMAT) and bone health, in the context of weight loss through caloric restriction or metabolic and bariatric surgery (MBS), in people living with obesity (PwO). We summarize the complex relationship between BMAT and bone health, provide an overview of noninvasive imaging techniques to quantify human BMAT, and discuss clinical studies measuring BMAT in PwO before and after weight loss. The relationship between BMAT and bone is subject to variations based on factors such as age, sex, menopausal status, skeletal sites, nutritional status, and metabolic conditions. The Bone Marrow Adiposity Society (BMAS) recommends standardizing imaging protocols to increase comparability across studies and sites, they have identified both water-fat imaging (WFI) and spectroscopy (1H-MRS) as accepted standards for in vivo quantification of BMAT. Clinical studies measuring BMAT in PwO are limited and have shown contradictory results. However, BMAT tends to be higher in patients with the highest visceral adiposity, and inverse associations between BMAT and bone mineral density (BMD) have been consistently found in PwO. Furthermore, BMAT levels tend to decrease after caloric restriction-induced weight loss. Although weight loss was associated with overall fat loss, a reduction in BMAT did not always follow the changes in fat volume in other tissues. The effects of MBS on BMAT are not consistent among the studies, which is at least partly related to the differences in the study population, skeletal site, and duration of the follow-up. Overall, gastric bypass appears to decrease BMAT, particularly in patients with diabetes and postmenopausal women, whereas sleeve gastrectomy appears to increase BMAT. More research is necessary to evaluate changes in BMAT and its connection to bone metabolism, either in PwO or in cases of weight loss through caloric restriction or MBS, to better understand the role of BMAT in this context and determine the local or systemic factors involved.

Omega-3 PUFAs prevent bone impairment and bone marrow adiposity in mouse model of obesity.

Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties.

Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics. Methods: BM donors (age: 48-85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed. Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2.

Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults' physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.

KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration.

Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cell-secreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions.

Osteoporosis and Bone Marrow Adipose Tissue.

PURPOSE OF REVIEW: This review focuses on the recent findings regarding bone marrow adipose tissue (BMAT) concerning bone health. We summarize the variations in BMAT in relation to age, sex, and skeletal sites, and provide an update on noninvasive imaging techniques to quantify human BMAT. Next, we discuss the role of BMAT in patients with osteoporosis and interventions that affect BMAT. RECENT FINDINGS: There are wide individual variations with region-specific fluctuation and age- and gender-specific differences in BMAT content and composition. The Bone Marrow Adiposity Society (BMAS) recommendations aim to standardize imaging protocols to increase comparability across studies and sites. Water-fat imaging (WFI) seems an accurate and efficient alternative for spectroscopy (1H-MRS). Most studies indicate that greater BMAT is associated with lower bone mineral density (BMD) and a higher prevalence of vertebral fractures. The proton density fat fraction (PDFF) and changes in lipid composition have been associated with an increased risk of fractures independently of BMD. Therefore, PDFF and lipid composition could potentially be future imaging biomarkers for assessing fracture risk. Evidence of the inhibitory effect of osteoporosis treatments on BMAT is still limited to a few randomized controlled trials. Moreover, results from the FRAME biopsy sub-study highlight contradictory findings on the effect of the sclerostin antibody romosozumab on BMAT. Further understanding of the role(s) of BMAT will provide insight into the pathogenesis of osteoporosis and may lead to targeted preventive and therapeutic strategies.

High-fat diet-induced obesity augments the deleterious effects of estrogen deficiency on bone: Evidence from ovariectomized mice.

Several epidemiological studies have suggested that obesity complicated with insulin resistance and type 2 diabetes exerts deleterious effects on the skeleton. While obesity coexists with estrogen deficiency in postmenopausal women, their combined effects on the skeleton are poorly studied. Thus, we investigated the impact of high-fat diet (HFD) on bone and metabolism of ovariectomized (OVX) female mice (C57BL/6J). OVX or sham operated mice were fed either HFD (60%fat) or normal diet (10%fat) for 12 weeks. HFD-OVX group exhibited pronounced increase in body weight (~86% in HFD and ~122% in HFD-OVX, p < 0.0005) and impaired glucose tolerance. Bone microCT-scanning revealed a pronounced decrease in trabecular bone volume/total volume (BV/TV) (-15.6 ± 0.48% in HFD and -37.5 ± 0.235% in HFD-OVX, p < 0.005) and expansion of bone marrow adipose tissue (BMAT; +60.7 ± 9.9% in HFD vs. +79.5 ± 5.86% in HFD-OVX, p < 0.005). Mechanistically, HFD-OVX treatment led to upregulation of genes markers of senescence, bone resorption, adipogenesis, inflammation, downregulation of gene markers of bone formation and bone development. Similarly, HFD-OVX treatment resulted in significant changes in bone tissue levels of purine/pyrimidine and Glutamate metabolisms, known to play a regulatory role in bone metabolism. Obesity and estrogen deficiency exert combined deleterious effects on bone resulting in accelerated cellular senescence, expansion of BMAT and impaired bone formation leading to decreased bone mass. Our results suggest that obesity may increase bone fragility in postmenopausal women.

The pathophysiology of osteoporosis in obesity and type 2 diabetes in aging women and men: The mechanisms and roles of increased bone marrow adiposity.

Osteoporosis is defined as a systemic skeletal disease characterized by decreased bone mass and micro-architectural deterioration leading to increased fracture risk. Osteoporosis incidence increases with age in both post-menopausal women and aging men. Among other important contributing factors to bone fragility observed in osteoporosis, that also affect the elderly population, are metabolic disturbances observed in obesity and Type 2 Diabetes (T2D). These metabolic complications are associated with impaired bone homeostasis and a higher fracture risk. Expansion of the Bone Marrow Adipose Tissue (BMAT), at the expense of decreased bone formation, is thought to be one of the key pathogenic mechanisms underlying osteoporosis and bone fragility in obesity and T2D. Our review provides a summary of mechanisms behind increased Bone Marrow Adiposity (BMA) during aging and highlights the pre-clinical and clinical studies connecting obesity and T2D, to BMA and bone fragility in aging osteoporotic women and men.

Novel thiazolidinedione analog reduces a negative impact on bone and mesenchymal stem cell properties in obese mice compared to classical thiazolidinediones.

OBJECTIVE: The use of thiazolidinediones (TZDs) as insulin sensitizers has been shown to have side effects including increased accumulation of bone marrow adipocytes (BMAds) associated with a higher fracture risk and bone loss. A novel TZD analog MSDC-0602K with low affinity to PPARγ has been developed to reduce adverse effects of TZD therapy. However, the effect of MSDC-0602K on bone phenotype and bone marrow mesenchymal stem cells (BM-MSCs) in relation to obesity has not been intensively studied yet. METHODS: Here, we investigated whether 8-week treatment with MSDC-0602K has a less detrimental effect on bone loss and BM-MSC properties in obese mice in comparison to first generation of TZDs, pioglitazone. Bone parameters (bone microstructure, bone marrow adiposity, bone strength) were examined by µCT and 3-point bending test. Primary BM-MSCs were isolated and measured for osteoblast and adipocyte differentiation. Cellular senescence, bioenergetic profiling, nutrient consumption and insulin signaling were also determined. RESULTS: The findings demonstrate that MSDC-0602K improved bone parameters along with increased proportion of smaller BMAds in tibia of obese mice when compared to pioglitazone. Further, primary BM-MSCs isolated from treated mice and human BM-MSCs revealed decreased adipocyte and higher osteoblast differentiation accompanied with less inflammatory and senescent phenotype induced by MSDC-0602K vs. pioglitazone. These changes were further reflected by increased glycolytic activity differently affecting glutamine and glucose cellular metabolism in MSDC-0602K-treated cells compared to pioglitazone, associated with higher osteogenesis. CONCLUSION: Our study provides novel insights into the action of MSDC-0602K in obese mice, characterized by the absence of detrimental effects on bone quality and BM-MSC metabolism when compared to classical TZDs and thus suggesting a potential therapeutical use of MSDC-0602K in both metabolic and bone diseases.

Next Generation Bone Marrow Adiposity Researchers: Report From the 1st BMAS Summer School 2021.

The first International Summer School on Bone Marrow Adiposity was organized by members of Bone Marrow Adiposity Society and held virtually on September 6-8 2021. The goal of this meeting was to bring together young scientists interested in learning about bone marrow adipose tissue biology and pathology. Fifty-two researchers from different backgrounds and fields, ranging from bone physiopathology to adipose tissue biology and hematology, participated in the summer school. The meeting featured three keynote lectures on the fundamentals of bone marrow adiposity, three scientific workshops on technical considerations in studying bone marrow adiposity, and six motivational and career development lectures, spanning from scientific writing to academic career progression. Moreover, twenty-one participants presented their work in the form of posters. In this report we highlight key moments and lessons learned from the event.

Impaired Bone Fracture Healing in Type 2 Diabetes Is Caused by Defective Functions of Skeletal Progenitor Cells.

The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high-fat diet and insulinopenia induced by treatment with streptozotocin, we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by -28.4 ± 7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06 ± 38.71%, and increased density of SCA1+ (+74.99 ± 29.19%) but not Runx2+ osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82 ± 33.05%), senescence gene signature (≈106.66 ± 34.03%), and LAMIN B1- senescent cell density (+225.18 ± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9 ± 6.2% which was inversely correlated with glucose levels (R2 = 0.48, P < .004) and callus adipose tissue area (R2 = .3711, P < .01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast-related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycemia per se and not just insulin levels is detrimental for bone healing.

Molecular differences of adipose-derived mesenchymal stem cells between non-responders and responders in treatment of transphincteric perianal fistulas.

BACKGROUND: Injection of autologous adipose tissue (AT) has recently been demonstrated to be an effective and safe treatment for anal fistulas. AT mesenchymal stem cells (AT-MSCs) mediate the healing process, but the relationship between molecular characteristics of AT-MSCs of the injected AT and fistula healing has not been adequately studied. Thus we aimed to characterize the molecular and functional properties of AT-MSCs isolated from autologous AT injected as a treatment of cryptogenic high transsphincteric perianal fistulas and correlate these findings to the healing process. METHODS: 27 patients (age 45 ± 2 years) diagnosed with perianal fistula were enrolled in the study and treated with autologous AT injected around the anal fistula tract. AT-MSCs were isolated for cellular and molecular analyses. The fistula healing was evaluated by MRI scanning after 6 months of treatment. AT-MSC phenotype was compared between responders and non-responders with respect to fistula healing. RESULTS: 52% of all patients exhibited clinical healing of the fistulas as evaluated 6 months after last injection. Cultured AT-MSCs in the responder group had a lower short-term proliferation rate and higher osteoblast differentiation potential compared to non-responder AT-MSCs. On the other hand, adipocyte differentiation potential of AT-MSCs was higher in non-responder group. Interestingly, AT-MSCs of responders exhibited lower expression of inflammatory and senescence associated genes such as IL1B, NFKB, CDKN2A, TPB3,TGFB1. CONCLUSION: Our data suggest that cellular quality of the injected AT-MSCs including cell proliferation, differentiation capacity and secretion of proinflammatory molecules may provide a possible mechanism underlying fistula healing. Furthermore, these biomarkers may be useful to predict a positive fistula healing outcome. TRIAL REGISTRATION: NTC04834609, Registered 6 April 2021. https://clinicaltrials.gov/ct2/show/NCT04834609.

Guidelines for Biobanking of Bone Marrow Adipose Tissue and Related Cell Types: Report of the Biobanking Working Group of the International Bone Marrow Adiposity Society.

Over the last two decades, increased interest of scientists to study bone marrow adiposity (BMA) in relation to bone and adipose tissue physiology has expanded the number of publications using different sources of bone marrow adipose tissue (BMAT). However, each source of BMAT has its limitations in the number of downstream analyses for which it can be used. Based on this increased scientific demand, the International Bone Marrow Adiposity Society (BMAS) established a Biobanking Working Group to identify the challenges of biobanking for human BMA-related samples and to develop guidelines to advance establishment of biobanks for BMA research. BMA is a young, growing field with increased interest among many diverse scientific communities. These bring new perspectives and important biological questions on how to improve and build an international community with biobank databases that can be used and shared all over the world. However, to create internationally accessible biobanks, several practical and legislative issues must be addressed to create a general ethical protocol used in all institutes, to allow for exchange of biological material internationally. In this position paper, the BMAS Biobanking Working Group describes similarities and differences of patient information (PIF) and consent forms from different institutes and addresses a possibility to create uniform documents for BMA biobanking purposes. Further, based on discussion among Working Group members, we report an overview of the current isolation protocols for human bone marrow adipocytes (BMAds) and bone marrow stromal cells (BMSCs, formerly mesenchymal), highlighting the specific points crucial for effective isolation. Although we remain far from a unified BMAd isolation protocol and PIF, we have summarized all of these important aspects, which are needed to build a BMA biobank. In conclusion, we believe that harmonizing isolation protocols and PIF globally will help to build international collaborations and improve the quality and interpretation of BMA research outcomes.

Bone marrow adipose tissue: Role in bone remodeling and energy metabolism.

Bone marrow adipose tissue (BMAT) has been considered for several decades as a silent bystander that fills empty space left in bone marrow following age-related decrease in hematopoiesis. However, recently new discoveries revealed BMAT as a secretory and metabolically active organ contributing to bone and whole-body energy metabolism. BMAT exhibits metabolic functions distinct from extramedullary adipose depots, relevant to its role in regulation of energy metabolism and its contribution to fracture risk observed in metabolic bone diseases. This review discusses novel insights of BMAT with particular emphasis on its contribution to the regulation of bone homeostasis. We also discuss the role of BMAT in regulation of fuel utilization and energy use that affect skeletal stem cell functions.