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.