Quantitative approaches to unravel bone marrow adipocyte site-specificity and its implication in hematopoiesis

Over the last decade, bone marrow (BM) adipose tissue (BMAT) has emerged as a distinct adipose depot with unique metabolic properties and effects both in homeostasis and in the progression of disease.

We provide a first comprehensive review of existing and emerging technologies including accompanying challenges for the study of bone marrow adipocytes (BMAds). The recommendations specified through this consensus opinion for standardization of methodological approaches are imperative on our quest to uncover the meaning, the sense, and the significance of BMAds. Due to its challenging localization within the bone and its fragile nature, imaging methods provide powerful tools for ex vivo or in vivo BMAT quantification as well as characterization through lipid profiling. While isolation methods are still evolving in the pursuit of the BMAd and its progenitor, lineage tracing and BMAT (environmental, pharmacological, or pathological) modulation studies have identified two sub-populations of BMAds dependent on skeletal location in the hematopoietic red or fatty yellow marrow.

We uncover changes to these compartments in the mouse skeleton with age or irradiation and bone marrow transplantation induced aplastic conversion of the marrow, through our novel imaging quantification tool for histological sections. Following this progression by magnetic resonance imaging in vivo emphasized a differential hematopoietic recovery within the long bones of mice. In order to delineate the subtle differences between BMAds of red and fatty marrow, we established first an adipocytic differentiation, and then a co-culture system to recapitulate the BM niche in vitro with the OP9 stromal cell line. Notably, Raman microspectroscopic analysis revealed nuances of lipid saturation levels previously reported in primary BMAds at the single droplet level that we propose is indicative of their maturation state. This may have implication for the documented differential hematopoietic support capacity of BMAds that we elucidate through co-culture with hematopoietic stem and progenitor cells. We were able to translate the established co-culture system onto a three-dimensional scaffold for construction of a tissue-engineered BM model. In combination with a novel approach for preparation of its in vivo injection, we have established the direct in vitro to in vivo transfer of a biomimetic BM niche.

The novel methodological approaches presented here to study BMAds and the hematopoietic microenvironment, have far-reaching applications for improving the outcome of BM disease modeling, biomolecular and drug screening, as well as in regenerative medicine at large.

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

The procedure-associated mortality rate of bone marrow transplantations (BMT) remains close to 25%. The BMT is therefore only indicated for life-threatening diseases, with no replacement of this technology to be expected in the foreseeable future. Modest improvements such as the use of G-CSF to accelerate hematopoietic recovery significantly reduce mortality. Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and are mainly quiescent, dividing only to self-renew. Via extracellular signals and interactions with other cells of the BM (microenvironment), a stable HSC pool is maintained, while hematopoietic progenitor cells (HPC) proliferate and differentiate to mature blood cell types. Marrow adipocytes (BMA) have recently been recognized as a hematopoietic regulatory entity, although the mechanisms by which it interacts with HSCs and HPCs (HSPC) in vivo, remain unknown. During the hematopoietic recovery period post-BMT, HSPCs increase their metabolism as a response to hematopoietic stress, partly modulated by the microenvironment. In this work we identified novel pharmacological approaches to accelerate hematopoietic recovery in the post-BMT period, addressed via two strategies: (i) by directly modulating HSC metabolism, and (ii) by regulating the differentiation of BMA. Increasing oxidative phosphorylation, combined with an impaired mitochondrial stress response, can severely compromise the regenerative capacity of HSCs. Here we demonstrate that the NAD+-boosting agent Nicotinamide Riboside (NR) reduces mitochondrial activity within HSCs through increased mitophagy, the recycling mechanism of damaged mitochondria. We observed in vitro NR treatment of HSCs to lead to increased asymmetric HSC divisions, which resulted in a significantly enlarged pool of progenitor cells, without HSC exhaustion. Dietary supplementation of NR to mice undergoing BMT accelerated blood recovery and improved survival. We therefore established a novel link between HSC mitochondrial stress, mitophagy and stem cell fate decision. Recent studies have suggested BMA maintain HSCs quiescent, which, may be detrimental to the increased expansion of HPCs in the recovery phase post-BMT. BM stromal cells (BMSC) are the cellular precursors of both adipocytes and osteoblasts, and they have been strongly implicated in supporting hematopoiesis by secreting cytokines such as SCF and Cxcl12. We observed that adipocytes quickly accumulate the marrow space of mice undergoing BMT, followed by an expansion hematopoiesis-supportive multipotent Sca1+/CD24+ stromal cells, coinciding with hematopoietic recovery. We developed a novel screening platform using Digital Holographic Microscopy (DHM), quantifying in vitro adipocytic differentiation non-invasively, allowing for follow-up co-cultures with HSPCs. Using the OP9 BMSC-line, we modulated adipocyte differentiation prior to co-culture using a pharmacological approach and observed that high adipocytic content was associated to a decrease in HPC expansion. We perfomed a screening of over 4000 FDA-approved drugs and natural compounds and identified one to efficiently prevent adipocytic differentiation and maintain the hematopoietic supportive capacity of OP9 stroma. Altogether, this work opens the door to alternative strategies accelerating hematopoietic reconstitution and unveils the potential to improve recovery of patients undergoing BMT.

EPFL2019

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

Chiheb Boussema, Olivier Burri, Nicolas Kunz, Vasco Ledebur de Antas de Campos, Olaia Maria Naveiras Torres-Quiroga, Shanti Rojas-Sutterlin, Rita Sarkis, Frédérica Schyrr, Daniel Naveed Tavakol, Josefine Catharina Pedersen Tratwal

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-mu CT) 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.MarrowQuantdiscerns 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 withMarrowQuantlies 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 ofin vivorecovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsiesMarrowQuantsuccessfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.

2020

New approaches to uncover the minimally functional hematopoietic niche using the adipocytic differentiation axis: pharmacological modulation and adrenal niche induction

Frédérica Schyrr

Hematopoietic Stem and Progenitor Cells (HSPCs) reside in their niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards differentiated cells. This highly plastic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal progenitor cells of which CXCL12-abundant reticular (CAR) cells have been best characterized. Adipocytes have been shown to inhibit hematopoietic progenitor proliferation, but pre-adipocytes can efficiently support the growth of hematopoietic cells in culture. This suggests that the same cellular lineage contains populations with divergent hematopoietic-supportive capacity.In the first part of this thesis, we hypothesized that pharmacological modulation of the adipocytic differentiation axis could inhibit bone marrow (BM)-derived adipocyte maturation and improve hematopoietic progenitor support. Using a phenotypic screen, we identified calcipotriol, a vitamin D derivative, as a strong inhibitor of adipocyte differentiation. In vivo administration of calcipotriol decreased BM adipocyte size at day 18 post BM transplantation, increased total number of colony-forming units (CFUs) per leg and improved the survival of Cxcl12 haploinsufficient mice. Thus, we identified this vitamin D derivative as our top candidate to accelerate hematopoietic recovery in radio/chemotherapy-induced aplasia by targeting the maturation of adipocytes in the BM.Then, we took advantage of a prospective cohort of patients to assess the association between bone health and hematopoietic output in the context of osteoporosis. Osteoporosis is characterized by reduced bone mineral density (BMD), microarchitectural deterioration and increased BM adipocytic content. We studied the association between BMD and differential blood counts. We found that significant associations between blood counts and BMD exist, but none was consistent across bone parameters or timepoints. This suggests that, for steady-state hematopoiesis, there is no clinically significant effect of the deteriorated bone microenvironment on blood production.In the last part, to further investigate the composition of the HSPC-supportive microenvironment, we explored the simpler, albeit functional, extramedullary niche. We showed that the adrenal gland can be transformed into a hematopoietic supportive environment and used as a model to study the minimal components of a de novo niche. This induced adrenal niche supports HSPCs, including serially transplantable hematopoietic stem cells. Circulating HSPCs home into the adrenal cortex, where they are in proximity to CAR cells. These findings are supported by the presence of CXCL12+ cells in human adrenal myelolipoma, a benign tumor that contains extramedullary hematopoietic tissue. We envision our model as a novel tool to study the minimal components needed for hematopoietic support.Thus, using a combination of clinical, translational and basic research, we discovered a new therapeutic candidate approach to improve hematopoietic support by using a vitamin D analog to modify the BM adiposity. We showed the resilience of the BM niche by observing no measurable effect of the osteoporotic niche on hematopoietic output in a large cohort of patients. Finally, we developed a model of de novo hematopoietic niche in the adrenal gland. Altogether, our work provides new strategies to improve our understanding of the hematopoietic niche.

EPFL2022