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

Abstract

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.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging

“…The similarities of the overall spectral fingerprint of EM2 with the spectra of cells in bone marrow in the intact trabecular bone section recorded in a different region of the bone (section LR2) can be seen in Supplementary Figure S11. Furthermore, a high agreement of EM2 with published spectra of bone marrow adipocytes can be found [43]. This assignment would also agree with the spatial distribution of EM2 (Figure 6b and Supplementary Figure S9).…”

MarrowDLD: a microfluidic method for label-free retrieval of fragile bone marrow cells

“…In addition to hematopoietic cells, it includes MKs and BMAds, both characterized by large size and fragility in suspension. To develop our sorting device, we first utilized OP9 cells, a line of bone marrow-derived mouse stromal cells known as a robust adipogenesis model [47], [48]. After in vitro adipocytic-induced differentiation, OP9 progenitors accumulated lipid droplets and underwent the expected size increase ( Fig.…”

“…2C-D depict the behavior of microbeads within MarrowDLD and the sorting workflow for adipocytic cultures. Figure 3A shows a sample of OP9-derived adipocytic culture (induced-OP9 [48]) transiting the MarrowDLD sorter at the inlet and outlet. Specifically, a single-cell suspension was continuously injected and focused using lateral sheath flows ( Fig.…”

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

“…In addition to hematopoietic cells, it includes MKs and BMAds, both characterized by their large size and fragility in suspension. To develop our sorting device, we first utilized OP9 cells, a non-clonal line of bone marrow-derived mouse stromal cells known as a robust adipogenesis model 47 , 48 . After in vitro adipocytic induced differentiation, OP9 progenitors accumulated lipid droplets and underwent the expected size increase (Fig.…”

“…Figure 2 C and 2 D depict the behavior of microbeads within MarrowCellDLD and the sorting workflow for adipocytic cultures. Figure 3 A shows a sample of OP9-derived adipocytic culture (induced-OP9 48 ), transiting the MarrowCellDLD sorter at the inlet and outlet. Specifically, a single-cell suspension was continuously injected and focused using lateral sheath flows (Fig.…”