Developing an in vitro model and quantitation method for bone marrow adiposity research
Widjaja, Vincensa Nicko (2021)
Widjaja, Vincensa Nicko
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Bone marrow adipocytes (BMAds) have long been considered a passive and pathological filler within the bone marrow (BM) microenvironment. Recent studies suggest that BMAds may actively influence bone and energy metabolism. As an emerging field, studies to address this discrepancy are hindered by methodological limitations, such as the lack of in vitro models and subjectivity in reporting BM adiposity. To that end, we developed a simple adipocyte differentiation model in vitro by exposing bone marrow stromal cells (BMSCs) to 10 µg/mL insulin- and 10^-6 M dexamethasone-containing medium. Adipogenesis was confirmed by the temporal increase in adipocyte-specific mRNA expression and typical morphology of adipocytes with intracellular lipid formation in vitro. Using a colour-based segmentation, we developed two image analysis workflows to report area-based adiposity (9.7%) and the proportion of adipocytes obtained by differentiation (13.2%) from our in vitro model. To quantitate and analyse BMAds from histological bone sections, we developed a semi-automated quantitation workflow. The algorithm is able to minimise false-positive detections from the area resembling BMAds, such as blood vessels and empty spaces. Our quantitation method is comparable to the manual approach (r^2= 0.92; p< 0.0001), with an average of 17% error detection rate, especially resulting from fused and object-overlapped adipocytes ex vivo. The preservation of BMAd morphology during sample processing may improve the accuracy of the quantitation method. From a 16-week old Sprague-Dawley rat, the median size of BMAds in vivo was 535.2 (361.4 – 792.3) µm^2. In the future, advancements from the in vitro model and quantitation methods would prospectively direct the establishment towards methodological standard in BM adiposity research.