Inducible CXCL12/CXCR4-dependent extramedullary hematopoietic niches in the adrenal gland.

Adult hematopoietic Stem and Progenitor Cells (HSPCs) reside in the bone marrow hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. While the complex bone marrow niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α (PDGFRα) expressing stromal nodules. We further show in CXCL12-GFP reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-Abundant Reticular (CAR) cells which compose the bone marrow HSPC niche. Mechanistically, HSPC homing to hormonally-induced adrenal glands was found dependent on the CXCR4/CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells co-expressing master niche-regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches which may pave the way to therapeutic applications.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells.

Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

Induction of mitochondrial recycling reverts age-associated decline of the hematopoietic and immune systems.

Aging compromises hematopoietic and immune system functions, making older adults especially susceptible to hematopoietic failure, infections and tumor development, and thus representing an important medical target for a broad range of diseases. During aging, hematopoietic stem cells (HSCs) lose their blood reconstitution capability and commit preferentially toward the myeloid lineage (myeloid bias)1,2. These processes are accompanied by an aberrant accumulation of mitochondria in HSCs3. The administration of the mitochondrial modulator urolithin A corrects mitochondrial function in HSCs and completely restores the blood reconstitution capability of 'old' HSCs. Moreover, urolithin A-supplemented food restores lymphoid compartments, boosts HSC function and improves the immune response against viral infection in old mice. Altogether our results demonstrate that boosting mitochondrial recycling reverts the aging phenotype in the hematopoietic and immune systems.

MarrowQuant 2.0: A Digital Pathology Workflow Assisting Bone Marrow Evaluation in Experimental and Clinical Hematology.

Bone marrow (BM) cellularity assessment is a crucial step in the evaluation of BM trephine biopsies for hematologic and nonhematologic disorders. Clinical assessment is based on a semiquantitative visual estimation of the hematopoietic and adipocytic components by hematopathologists, which does not provide quantitative information on other stromal compartments. In this study, we developed and validated MarrowQuant 2.0, an efficient, user-friendly digital hematopathology workflow integrated within QuPath software, which serves as BM quantifier for 5 mutually exclusive compartments (bone, hematopoietic, adipocytic, and interstitial/microvasculature areas and other) and derives the cellularity of human BM trephine biopsies. Instance segmentation of individual adipocytes is realized through the adaptation of the machine-learning-based algorithm StarDist. We calculated BM compartments and adipocyte size distributions of hematoxylin and eosin images obtained from 250 bone specimens, from control subjects and patients with acute myeloid leukemia or myelodysplastic syndrome, at diagnosis and follow-up, and measured the agreement of cellularity estimates by MarrowQuant 2.0 against visual scores from 4 hematopathologists. The algorithm was capable of robust BM compartment segmentation with an average mask accuracy of 86%, maximal for bone (99%), hematopoietic (92%), and adipocyte (98%) areas. MarrowQuant 2.0 cellularity score and hematopathologist estimations were highly correlated (R2 = 0.92-0.98, intraclass correlation coefficient [ICC] = 0.98; interobserver ICC = 0.96). BM compartment segmentation quantitatively confirmed the reciprocity of the hematopoietic and adipocytic compartments. MarrowQuant 2.0 performance was additionally tested for cellularity assessment of specimens prospectively collected from clinical routine diagnosis. After special consideration for the choice of the cellularity equation in specimens with expanded stroma, performance was similar in this setting (R2 = 0.86, n = 42). Thus, we conclude that these validation experiments establish MarrowQuant 2.0 as a reliable tool for BM cellularity assessment. We expect this workflow will serve as a clinical research tool to explore novel biomarkers related to BM stromal components and may contribute to further validation of future digitalized diagnostic hematopathology workstreams.

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

[Short telomere syndrome in adults: a rare entity that should be evoked]. / Syndrome des télomères courts chez l'adulte†: une entité rare qu'il faut savoir évoquer.

Short telomere syndrome (STS) is a group of rare, often underrecognized, diseases caused by defects in telomere-maintenance genes, leading to abnormal telomere shortening and associated with diverse multi-organ manifestations. In pediatric patients, STS typically presents with mucocutaneous or gastrointestinal lesions, bone marrow failure and neoplasia. In adulthood, aplastic bone marrow disease, liver disease and pulmonary fibrosis are classic clinical manifestations. At present, medical treatment options for STS remain limited. Danazol, a synthetic androgenic hormone, can slow down telomere shortening and thus limit the progression of the disease. Finally, hematopoietic, hepatic and pulmonary transplantation, sometimes combined, may be discussed in a multidisciplinary setting in certain situations.

Le syndrome des télomères courts (STC) est un groupe de maladies rares dues à un défaut dans les gènes de maintenance des télomères, provoquant leur raccourcissement anormal et des manifestations cliniques multiorganiques. Dans l'enfance, le STC se présente par des lésions mucocutanées et gastro-intestinales, une insuffisance médullaire et des néoplasies. À l'âge adulte, une atteinte médullaire aplasiante, hépatique, et une fibrose pulmonaire sont des manifestations cliniques classiques. Les options thérapeutiques pour le STC restent limitées. Le danazol, une hormone androgène synthétique, permet, parfois, de freiner le raccourcissement télomérique et de limiter la progression de la maladie. Finalement, les transplantations hématopoïétique, hépatique et pulmonaire sont discutées dans certaines situations de manière multidisciplinaire.

Differential Blood Counts Do Not Consistently Predict Clinical Measurements of Bone Mineral Density and Microarchitecture at Homeostasis.

The hematopoietic stem cell niche constitutes a complex bone marrow (BM) microenvironment. Osteoporosis is characterized by both reduced bone mineral density (BMD) and microarchitectural deterioration, constituting the most frequent alteration of the BM microenvironment. It is unclear to which extent modifications of the BM microenvironment, including in the context of osteoporosis, influence blood cell production. We aimed to describe the association between lumbar spine and total hip BMD and microarchitecture (assessed by trabecular bone score [TBS]) and differential blood counts. Data were collected at two time points from 803 (first assessment) and 901 (second assessment) postmenopausal women participating in the CoLaus/OsteoLaus cohort, a population-based sample in Lausanne, Switzerland. Participants with other active disease or treatment that could influence hematopoiesis or osteoporosis were excluded. Bivariate and multivariate associations between each peripheral blood cell count and BMD or TBS were performed. Additionally, participants in the highest BMD and TBS tertiles were compared with participants in the lowest BMD and TBS tertiles. At first assessment, only neutrophils were significantly different in the lowest BMD and TBS tertile (3.18 ± 0.09 versus 3.47 ± 0.08 G/L, p = 0.028). At the second assessment, leucocytes (5.90 ± 0.11 versus 5.56 ± 0.10 G/L, p = 0.033), lymphocytes (1.87 ± 0.04 versus 1.72 ± 0.04 G/L p = 0.033), and monocytes (0.49 ± 0.01 versus 0.46 ± 0.1 G/L, p = 0.033) were significantly different. Power analysis did not identify quasi-significant associations missed due to sample size. Although significant associations between blood counts and BMD or TBS were found, none was consistent across bone measurements or assessments. This study suggests that, at homeostasis and in postmenopausal women, there is no clinically significant association between the osteoporotic microenvironment and blood production output as measured by differential blood counts. In the context of conflicting reports on the relationship between osteoporosis and hematopoiesis, our study represents the first prospective two time-point analysis of a large, homogenous cohort at steady state. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cryogel-based Injectable 3D Microcarrier Co-culture for Support of Hematopoietic Progenitor Niches.

Although hematopoietic stem cell (HSC) transplantation can restore functional hematopoiesis upon immune or chemotherapy-induced bone marrow failure, complications often arise during recovery, leading to up to 25% transplant-related mortality in treated patients. In hematopoietic homeostasis and regeneration, HSCs in the bone marrow give rise to the entirety of cellular blood components. One of the challenges in studying hematopoiesis is the ability to successfully mimic the relationship between the stroma and hematopoietic stem and progenitor cells (HSPCs). This study and the described protocols propose an advantageous method for culturing and assessing stromal hematopoietic support in three dimensions, representing a simplified in vitro model of the bone marrow niche that can be transplanted in vivo by injection. By co-culturing OP9 bone marrow-derived stromal cells (BMSCs) and cKit+ Sca-1+ Lin- (KLS+ ) HSPCs on collagen-coated carboxymethylcellulose scaffolds for 2 weeks in the absence of cytokines, we established a methodology for in vivo subcutaneous transplantation. With this model we were able to detect early signs of extramedullary hematopoiesis. This work can be useful for studying various stromal cell populations in co-culture, as well as simple transfer by injection of these scaffolds in vivo for heterotopic regeneration of the marrow microenvironment. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of HSPCs from mice Basic Protocol 2: Co-seeding of HSPCs and BMSCs on collagen-coated CCMs Basic Protocol 3: Maintenance, real-time imaging, and analysis of co-seeded scaffolds Basic Protocol 4: End-point analysis of co-seeded scaffolds using flow cytometry and CFU assays Basic Protocol 5: Transplantation of scaffolds by subcutaneous injection Support Protocol: Preparation of custom scaffold drying device.

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.

An Injectable Meta-Biomaterial: From Design and Simulation to In Vivo Shaping and Tissue Induction.

A novel type of injectable biomaterial with an elastic softening transition is described. The material enables in vivo shaping, followed by induction of 3D stable vascularized tissue. The synthesis of the injectable meta-biomaterial is instructed by extensive numerical simulation as a suspension of irregularly fragmented, highly porous sponge-like microgels. The irregular particle shape dramatically enhances yield strain for in vivo stability against deformation. Porosity of the particles, along with friction between internal surfaces, provides the elastic softening transition. This emergent metamaterial property enables the material to reversibly change stiffness during deformation, allowing native tissue properties to be matched over a wide range of deformation amplitudes. After subcutaneous injection in mice, predetermined shapes can be sculpted manually. The 3D shape is maintained during excellent host tissue integration, with induction of vascular connective tissue that persists to the end of one-year follow-up. The geometrical design is compatible with many hydrogel materials, including cell-adhesion motives for cell transplantation. The injectable meta-biomaterial therefore provides new perspectives in soft tissue engineering and regenerative medicine.

Bone marrow adiposity and the hematopoietic niche: A historical perspective of reciprocity, heterogeneity, and lineage commitment.

PURPOSE: Here we review the current knowledge on bone marrow adipocytes (BMAds) as active contributors to the regulation of the hematopoietic niche, and as potentially pivotal players in the progression of hematological malignancies. We highlight the hierarchical and functional heterogeneity of the adipocyte lineage within the bone marrow, and how potentially different contexts dictate their interactions with hematopoietic populations. RECENT FINDINGS: Growing evidence associates the adipocyte lineage with important functions in hematopoietic regulation within the BM niche. Initially proposed to serve as negative regulators of the hematopoietic microenvironment, studies have also demonstrated that BMAds positively influence the survival and maintenance of hematopoietic stem cells (HSCs). These seemingly incongruous findings may at least be partially explained by stage-specificity across the adipocytic differentiation axis and by BMAds subtypes, suggesting that the heterogeneity of these populations allows for differential context-based interactions. One such distinction relies on the location of adipocytes. Constitutive bone marrow adipose tissue (cBMAT) historically associates to the "yellow" marrow containing so-called "stable" BMAs larger in size, less responsive to stimuli, and linked to HSC quiescence. On the other hand, regulated bone marrow adipose tissue (rBMAT)-associated adipocytes, also referred to as "labile" are smaller, more responsive to hematopoietic demand and strategically situated in hematopoietically active regions of the skeleton. Here we propose a model where the effect of distinct BM stromal cell populations (BMSC) in hematopoiesis is structured along the BMSC-BMAd differentiation axis, and where the effects on HSC maintenance versus hematopoietic proliferation are segregated. In doing so, it is possible to explain how recently identified, adipocyte-primed leptin receptor-expressing, CXCL12-high adventitial reticular cells (AdipoCARs) and marrow adipose lineage precursor cells (MALPs) best support active hematopoietic cell proliferation, while adipose progenitor cells (APCs) and maturing BMAd gradually lose the capacity to support active hematopoiesis, favoring HSC quiescence. Implicated soluble mediators include MCP-1, PAI-1, NRP1, possibly DPP4 and limiting availability of CXCL12 and SCF. How remodeling occurs within the BMSC-BMAd differentiation axis is yet to be elucidated and will likely unravel a three-way regulation of the hematopoietic, bone, and adipocytic compartments orchestrated by vascular elements. The interaction of malignant hematopoietic cells with BMAds is precisely contributing to unravel specific mechanisms of remodeling. SUMMARY: BMAds are important operative components of the hematopoietic microenvironment. Their heterogeneity directs their ability to exert a range of regulatory capacities in a manner dependent on their hierarchical, spatial, and biological context. This complexity highlights the importance of (i) developing experimental tools and nomenclature adapted to address stage-specificity and heterogeneity across the BMSC-BMAd differentiation axis when reporting effects in hematopoiesis, (ii) interpreting gene reporter studies within this framework, and (iii) quantifying changes in all three compartments (hematopoiesis, adiposity and bone) when addressing interdependency.

Acquired haemophilia A in the postpartum and risk of relapse in subsequent pregnancies: A systematic literature review.

BACKGROUND: About 1%-5% of acquired haemophilia A cases affect mothers in the postpartum setting. AIMS: This study delineates the characteristics of this disease, specific to the postpartum setting, notably relapse in subsequent pregnancies. METHODS: Report of two cases and literature study (1946-2019), yielding 73 articles describing 174 cases (total 176 cases). RESULTS: Patients were aged 29.9 years (17-41) and 69% primigravidae. Diagnosis was made at a median of 60 days after delivery (range 0-308). Bleeding types were obstetrical (43.4%), cutaneous (41.3%), and muscular (36.7%). In >90% of the cases, FVIII at diagnosis was <1% (range 0%-8%). FVIII inhibitor was documented in 75.4% cases (median titre of 20 BU/ml, range 1-621). Haemostatic treatment was necessary in 57.1% using fresh frozen plasma (16%), factor concentrate (27.6%) and/or bypassing agents (37.4%). Immunosuppressive treatment was administered in 90.8%, mostly steroids (85.3%), alone or combined with immunosuppressants (27%). Rituximab was used mostly as a second line treatment. Only 24 patients (13.6%) had documented subsequent pregnancies and 6 (22.2%) suffered haemophilia recurrence during pregnancy. CONCLUSION: This study allows better definition of: (1) clinical and laboratory characteristics of postpartum acquired haemophilia, (2) response to therapy, and (3) the risk of relapse for subsequent pregnancies.

MarrowQuant Across Aging and Aplasia: A Digital Pathology Workflow for Quantification of Bone Marrow Compartments in Histological Sections.

The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-µCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, MarrowQuant, which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. MarrowQuant discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, AdipoQuant, fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with MarrowQuant lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of in vivo recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies MarrowQuant successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.

Smart Hydrogels for the Augmentation of Bone Regeneration by Endogenous Mesenchymal Progenitor Cell Recruitment.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose.