Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment

Abstract

Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

Fig. 1

Six culture configurations were evaluated in this study. (A) Cells were cultured in mono-, co-, or tri-culture hydrogel constructs for up to 7 days and then analyzed as depicted above. After one day in culture, media was changed and half of the constructs were switched to high glucose. Shades of gray and white blocks represent hydrogel blocks of different types of cells in either mono-, co-, or tri-culture. (B) Tri-culture configurations consisted of adipocytes, MSCs, and osteoblasts (designated OMA); co-culture configurations consisted of osteoblasts and MSCs or adipocytes and MSCs (designated OMO and AMA, respectively); and mono-culture configurations consisted of osteoblasts, MSCs or adipocytes (designated OOO, MMM, and AAA, respectively).

Fig. 2

Adipocytes were assessed for response to culture conditions. (A) Representative images of adipocytes stained with Nile Red, specific for lipids (green) and counterstained with Hoechst, specific for nuclei (blue). (B) PLS-DA was used to build models for adipocytes assigned to classes by culture configuration. Models yielded two latent variables that discriminated adipocytes by culture configuration (R²Y=0.93 and Q²= 0.92). (C) For AAA culture, classes based on normal and high glucose levels were discriminated using PLS-DA (AAA: R²Y= 0.79 and Q²= 0.70). The corresponding weight plot can be found in the supplementary information (Fig. S1C). In all models, dashed lines represent the 95% confidence limit of the distribution of weights.

Fig. 3

Viability of adipocytes was assessed in each culture configuration at normal and high glucose levels. Fraction viable cells (via LIVE/DEAD staining) for each culture condition is presented (n = 3 for AAA, AMA; n ≥2 for OMA; no statistically significant differences were seen, p < 0.05).

Fig. 4

Osteoblasts were assessed for response to culture conditions. (A) Representative images of osteoblasts stained for ALP, a marker of osteogenic differentiation (red), and counterstained with Hoechst for nuclei (blue). Noticeably higher amount of ALP production was seen in osteoblasts from OMA cultures at day 1. (B) PCA was applied to the gene expression data of the global osteoblast population, yielding two principal components (R²X= 0.81 and Q²= 0.56). (C) For OOO and OMA cultures, classes based on normal and high glucose levels were discriminated using PLS-DA (OOO: R²Y= 0.5 and Q²= 0.43; OMA: R²Y=0.62 and Q²= 0.55). The corresponding weight plots can be found in the supplementary information (Fig. S3B). In all models, dashed lines represent the 95% confidence limit of the distribution of scores.

Fig. 5

Osteoblast viability was measured in response to culture conditions. Fraction viable cells (via LIVE/DEAD staining) for each culture condition is presented (n = 3 for OOO, OMO; n ≥2 for OMA;* = Significantly different from same culture configuration and glucose condition on Day 1; p < 0.05).

Fig. 6

MSCs were assessed for gene expression changes response to culture conditions. PLS-DA models were constructed for MSCs assigned to classes by culture configuration. Models yielded three latent variables that discriminated MSCs by culture configuration (R²Y=0.73 and Q²= 0.63). In all models, dashed lines represent the 95% confidence limit of the distribution of weights.

Fig. 7

MSC clonogenicity was measured post-culture using colony forming assays (A) Number of colony-forming units greater than 2 mm in diameter per dish (n = 3) (* = Significantly different from same culture configuration; p < 0.05). (B) MSC viability was measured in response to culture conditions. Fraction viable cells (via LIVE/DEAD staining) for each culture condition is presented (n = 3 for MMM, OMO, AMA; n ≥2 for OMA; * = Significantly different from same culture configuration and glucose condition on Day 1; p < 0.05).