MarrowQuantAcross 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-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.

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

Josefine Catharina Pedersen Tratwal

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.

EPFL2020

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

The role of MYC in bone and in the hematopoietic stem cell niche

Maike Jaworski

The nuclear protein c-Myc is one of the most potent proto-oncogenes described. Its expression is found to be deregulated in more than 50 percent of human cancers, where it has been shown to control a number of biological processes including driving proliferation, cell growth and angiogenesis while inhibiting differentiation. The effects of c-Myc are highly cell type specific and include cell autonomous and non-cell autonomous processes. In order to examine the effect of excess amounts of c-Myc in bone, we have generated a mouse line expressing the tTA transactivator under the control of the mouse 2.3 kb col1a1 promoter (named Col1a1::tTA), which directs transgene expression to mature osteoblasts. By combining this transgene with the Tet-O-Myc allele, we generated a Col1a1::tTA; Tet-O-Myc double transgenic mouse model in which it is possible to inducibly express human c-MYC in osteoblasts during development and in the adult. We document that the used 'tet-off' system directs transgene expression to bones and that c-MYC can be completely repressed by treatment with doxycycline. Using this mouse model, we demonstrate that overexpression of c-MYC leads to remarkable changes in mutant bones, which depend greatly in their severity on the developmental time point of c-MYC induction. Unrestricted c-MYC expression, starting with the first emergence of mature osteoblasts in the fetal skeleton (∼E14.0), leads to lethality of the mutants at birth; their bones are shortened and thicker than in controls. Later induction of c-MYC allows survival, but leads to significant changes in bone morphology. Mutant bones develop a very irregular bone structure with signs of excessive bone formation. Surprisingly, this also correlates with definite signs of excessive osteoclast activity, which explains the formation of cavities seen in the bone matrix. Osteoblasts not only produce bone matrix, they are also very important niche cells for hematopoietic cells, especially for hematopoietic stem cells and B cells. We find that c-MYC overexpression severely impairs B lymphopoiesis and also observe signs of extramedullary hematopoiesis in the spleen, which is indicative of a compromised HSC environment in the bone marrow. Most strikingly, these animals develop frank osteosarcomas leading to a much reduced life span. Tumors arise in various bones, with the ribs and hips being the most prominent sites. These c-MYC driven tumors are very aggressive as is evident by their capacity to spontaneously metastasize to lung and liver, a rather rare phenomenon in mouse models, indicating that these tumors share important aspects of human osteosarcomas. Molecular analysis of c-MYC overexpressing osteoblasts has revealed a defect in their terminal differentiation program, rendering them incapable to progress from a relatively immature proliferative to a quiescent, calcified matrix producing state. To our knowledge this is the first report of experimental osteosarcoma formation, originating from a mature and lineage committed osteoblast population indicating that c-MYC may be able to de-differentiate more mature cells. In summary, our novel conditional mouse model showed that c-MYC expression in mature osteoblasts can lead to the formation of highly aggressive and metastatic osteosarcomas, a lethal disease in humans. This model will be the basis for further studies elucidating the molecular effects of c-MYC on de-differentiation and metastasis formation, processes, which remain poorly understood but are critical events in highly malignant cancers.

EPFL2009