Development of a 3D bone marrow adipose tissue model

Abstract

Over the past twenty years, evidence has accumulated that biochemically and spatially defined networks of extracellular matrix, cellular components, and interactions dictate cellular differentiation, proliferation, and function in a variety of tissue and diseases. Modeling in vivo systems in vitro has been undeniably necessary, but when simplified 2D conditions rather than 3D in vitro models are used, the reliability and usefulness of the data derived from these models decreases. Thus, there is a pressing need to develop and validate reliable in vitro models to reproduce specific tissue-like structures and mimic functions and responses of cells in a more realistic manner for both drug screening/disease modeling and tissue regeneration applications. In adipose biology and cancer research, these models serve as physiologically relevant 3D platforms to bridge the divide between 2D cultures and in vivo models, bringing about more reliable and translationally useful data to accelerate benchtop to bedside research. Currently, no model has been developed for bone marrow adipose tissue (BMAT), a novel adipose depot that has previously been overlooked as "filler tissue" but has more recently been recognized as endocrine-signaling and systemically relevant. Herein we describe the development of the first 3D, BMAT model derived from either human or mouse bone marrow (BM) mesenchymal stromal cells (MSCs). We found that BMAT models can be stably cultured for at least 3 months in vitro, and that myeloma cells (5TGM1, OPM2 and MM1S cells) can be cultured on these for at least 2 weeks. Upon tumor cell co-culture, delipidation occurred in BMAT adipocytes, suggesting a bidirectional relationship between these two important cell types in the malignant BM niche. Overall, our studies suggest that 3D BMAT represents a "healthier," more realistic tissue model that may be useful for elucidating the effects of MAT on tumor cells, and tumor cells on MAT, to identify novel therapeutic targets. In addition, proteomic characterization as well as microarray data (expression of >22,000 genes) coupled with KEGG pathway analysis and gene set expression analysis (GSEA) supported our development of less-inflammatory 3D BMAT compared to 2D culture. In sum, we developed the first 3D, tissue-engineered bone marrow adipose tissue model, which is a versatile, novel model that can be used to study numerous diseases and biological processes involved with the bone marrow.

Keywords: 3D; Bone marrow adipose; Multiple myeloma; Silk scaffolds; Tissue engineering.

Figure 1. Development and Characterization of 3D Mouse Bone Marrow Adipose Tissue Derived from Whole Bone Marrow using Confocal Microscopy

A) C3H mouse whole BM was seeded onto silk scaffolds and confocal imaged at day 3, 7, 14, 21, and 26. Scaffold is autofluorescent in every channel and appears purple/pink. A–C) Live dead imaging shows live cells are green with calcein AM stain and dead cells are red with ethidium homodimer-1. A) Day 3 of culture: most cells are alive (green) with a round morphology. B) Day 7 of culture: live cells have an elongated, mesenchymal morphology rather than rounded, and they are located along the scaffold. C) Day 14 culture (day 4 adipogenic differentiation) live-dead imaging. More fibroblastic-like and fewer round cells were observed. Yellow/orange spheres indicate autofluorescence from lipids. D–F). Fixed scaffolds stained with Oil Red O (lipids, red), phalloidin (actin, green), and DAPI (nuclei, blue). Scaffold is autofluorescent (maroon). Both adipocytes and undifferentiated stromal cells are observed throughout the scaffold. D) Day 21 culture, day 11 adipogenesis. E, F) Day 26 culture, day 16 adipogenesis. Scale bar =100 μm. White arrows indicate scaffold; blue arrows indicate dead cells; yellow arrows indicate stromal cells; asterisks indicate adipocytes.

Figure 2. Development and Characterization of 3D Human Bone Marrow Adipose Tissue Derived from hMSCs

A–B) Live dead imaging shows live cells are green with calcein AM stain and dead cells are red with ethidium homodimer-1, (A) Day 1 and (B) Day 9 (day 8 of adipogenesis) (B) after hMSC seeding. C–D) Fixed scaffolds stained with Oil Red O (lipids, red), phalloidin (actin, green), and DAPI (nuclei, blue). Scaffold is autofluorescent (red or maroon). Both adipocytes and undifferentiated stromal cells are observed throughout the scaffold. C) Day 10 (day 9 of adipogenesis). D) Day 28 (day 27 of adipogenesis). Scale bar = 200 μm. Representative images from n=4 different hMSC donors. White arrows indicate scaffold; blue arrows indicate dead cells; yellow arrows indicate stromal cells; asterisks indicate adipocytes.

Figure 3. Microarray Gene Expression Analysis of 2D versus 3D Mouse BMAT

A) Principal component analysis (PCA), a multivariate, unbiased analysis, demonstrates distinct grouping of samples from 2D (red) and 3D (blue) mBMAT. PC1 correlated with culture conditions (3D vs 2D) demonstrating the significant impact of 3D culture. B) Microarray heat map showing hierarchical clustering of the 81 genes that passed the FDR<0.01 and fold change>2 filter. The heat map color scale is reflective of the standardized normalized expression data input, which was standardized to a mean of zero and a standard deviation of one. Red indicates overexpression and green indicates underexpression.

Figure 4. Proteomic Analysis of 3D vs 2D BMAT

Mass spectrometry and proteomic analysis was performed on 2D and 3D mBMAT. Normalized Principal Component (PC) Analysis (PCA) (PC 1 vs PC 2, and PC 1 vs PC 3) of 2D and 3D BMAT samples versus primary adipose depots (GWAT, gonadal white adipose tissue, and BAT, brown adipose tissue) demonstrates distinct grouping based on adipose origin and also a distinction between 3D and 2D BMAT. N ≥ 3 scaffolds or wells were combined together per biological replicate and n=4 biological replicates (individual mice) were used. N=5 technical repeats per sample were used. D1 represents PC 1, D2 represents PC 2, and D3 represents PC 3.

Figure 5. Human BMAT Co-Culture Supports MM1S Myeloma Cells

Maximum projections of confocal imaging of co-cultures. hBMAT was seeded to silk, cultured until confluent and then switched to adipogenic media for 37 days. Then scaffolds were switched to a co-culture media and seeded with GFP⁺/Luc⁺MM1S cells and imaged at 1 week (A), 2 weeks (B) or 3 weeks (C). Fixed scaffolds were stained with Oil Red O (lipids, red), phalloidin (actin, green), and DAPI (nuclei, blue). Scaffold is autofluorescent (maroon). Both adipocytes and undifferentiated stromal cells are observed throughout the BMAT and negatively impacted by myeloma cells. Scale bar = 100 μm. White arrows indicate tumor cells; yellow arrows indicate stromal cells; asterisks indicate adipocytes.

Figure 6. mBMAT and mMSC Co-Cultures Support 5TGM1 and MM1S Myeloma Cells

Maximum projections of confocal imaging of scaffolds fixed and stained with Oil Red O (lipids, red), phalloidin (actin, green), and DAPI (nuclei, blue). Scaffold is autofluorescent maroon/blue. A) mMSCs and mBMAT (derived from C57BL/6J mMSCs expanded in vitro and seeded to scaffolds) after 1 week culture or co-cultures with GFP⁺/Luc⁺5TGM1. Lipid content and stroma cell count appeared decreased in BMAT when co-cultured with myeloma cells. B) Quantification of BMAT adipocyte diameter after 2 week culture with GFP⁺/Luc⁺5TGM1 or GFP⁺/Luc⁺MM1S in co-culture or diluted media. Data represented as mean ± SEM, n=9 (n=3 mice per group, n=3 scaffolds per mouse). C) C57BL6/KaLwRij-derived MSC differentiation into 3D BMAT after 11 days of adipogenesis and cultured alone (Top) or co-cultured with GFP⁺/Luc⁺5TGM1 cells (Bottom) for 1 week. Interestingly, some myeloma cells contained small lipid droplets (yellow arrows), but most did not (white arrow). Representative images from n=2 mice with n=6 scaffolds per mouse for monoculture and co-culture. White scale bar = 200 μm; yellow scale bar = 50 μm.