Three-dimensional in vitro tri-culture platform to investigate effects of crosstalk between mesenchymal stem cells, osteoblasts, and adipocytes

Abstract

The bone marrow niche for mesenchymal stem cells (MSCs) contains different amounts of bone and fat that vary with age and certain pathologies. How this dynamic niche environment may affect their differentiation potential and/or healing properties for clinical applications remains unknown, largely due to the lack of physiologically relevant in vitro models. We developed an enabling platform to isolate and study effects of signaling interactions between tissue-scale, laminated hydrogel modules of multiple cell types in tandem. We applied this platform to co- and tri-culture of primary human MSCs, osteoblasts, and adipocytes over 18 days in vitro. Each cell type was analyzed separately with quantitative polymerase chain reaction (qPCR) and histochemistry for several mesenchymal lineage markers. Distinct expression dynamics for osteogenic, adipogenic, chondrogenic, and myogenic transcriptional regulators resulted within each cell type depending on its culture setting. Incorporating this data into multivariate models produced latent identifiers of each emergent cell type dependent on its co- or tri-culture setting. Histological staining showed sustained triglyceride storage in adipocytes regardless of culture condition, but transient alkaline phosphatase activity in both osteoblasts and MSCs. Taken together, our results suggest novel emergent phenotypes for MSCs, osteoblasts, and adipocytes in bone marrow that are dependent on and result in part from paracrine interactions with their neighboring cell types.

FIG. 1.

Sample fabrication and study design. Fabrication of co- and tri-culture constructs using the techniques outlined in the Materials and Methods section yields sample sets with well-segregated cell populations [photographs: sample tri-laminated hydrogel construct after reaching equilibrium swelling (left), and confocal image demonstrating human MSCs differentially stained with CellTracker Green (bottom) or Orange (top) segregated at an interface between modules]. Three sample types were examined in this study: MSCs in the center module flanked by only one other cell type [co-culture controls: osteoblasts (OMO) or adipocytes (AMA)] or by both cell types (tri-culture; OMA). MSCs, mesenchymal stem cells. Color images available online at www.liebertonline.com/tea

FIG. 2.

Co-culture and tri-culture differentially affect expression dynamics of osteoblastic (RUNX2 and Osteocalcin) and adipocytic (PPARγ2 and Leptin) genes in adipocytes but does not affect triglyceride storage with time. (A) Adipocyte expression levels of gene regulators of several mesenchymal lineages relative to RPS18 and ACTB over 18 days in co- and tri-culture. Values scaled×10³. *Significantly different from another day, same culture type; ^#significantly different from another culture type, same day; p<0.05. (B) Oil Red O staining of triglyceride storage vesicles (arrows) in adipocytes from AMA and OMA culture conditions over time (brightfield microscopy; scale bar=50 μm, inset scale bar=20 μm). Color images available online at www.liebertonline.com/tea

FIG. 3.

Co-culture and tri-culture differentially affect expression dynamics of osteoblastic (RUNX2 and Osteocalcin) and chondrogenic (SOX9) genes in osteoblasts, in addition to persistence of alkaline phosphatase activity over time. (A) Osteoblast expression levels of gene regulators of several mesenchymal lineages relative to RPS18 and ACTB over 18 days in co- and tri-culture. Values scaled×10³. *Significantly different from another day, same culture type; ^#significantly different from another culture type, same day; p<0.05. (B) In situ alkaline phosphatase substrate conversion in osteoblasts from OMO and OMA culture conditions over time (scale bar=20 μm; arrows indicate cells with enzyme activity). Color images available online at www.liebertonline.com/tea

FIG. 4.

Co- and tri-culture differentially affect expression levels and dynamics of several lineage-specific transcription factors (but not terminal differentiation markers) in MSCs, while only causing scant and transient alkaline phosphatase expression in MSCs from osteoblast-containing cultures. (A) MSC expression levels of gene regulators of several mesenchymal lineages relative to RPS18 and ACTB over 18 days in co- and tri-culture. Values scaled×10³. *Significantly different from another day, same culture type; ^#significantly different from another culture type, same day; p<0.05. (B) Absence of Oil Red O staining in MSCs (arrows) from different co- and tri-culture conditions over time (brightfield microscopy; scale bar=50 μm; inset scale bar=20 μm). (C) In situ alkaline phosphatase substrate conversion in MSCs from different co- and tri-culture conditions over time (scale bar=20 μm; arrows indicate cells with enzyme activity). Color images available online at www.liebertonline.com/tea

FIG. 4.

Co- and tri-culture differentially affect expression levels and dynamics of several lineage-specific transcription factors (but not terminal differentiation markers) in MSCs, while only causing scant and transient alkaline phosphatase expression in MSCs from osteoblast-containing cultures. (A) MSC expression levels of gene regulators of several mesenchymal lineages relative to RPS18 and ACTB over 18 days in co- and tri-culture. Values scaled×10³. *Significantly different from another day, same culture type; ^#significantly different from another culture type, same day; p<0.05. (B) Absence of Oil Red O staining in MSCs (arrows) from different co- and tri-culture conditions over time (brightfield microscopy; scale bar=50 μm; inset scale bar=20 μm). (C) In situ alkaline phosphatase substrate conversion in MSCs from different co- and tri-culture conditions over time (scale bar=20 μm; arrows indicate cells with enzyme activity). Color images available online at www.liebertonline.com/tea

FIG. 5.

Statistical modeling based on covariance of the expression of several mesenchymal lineage genes yields two latent variables that are able to distinguish MSCs from adipocytes and osteoblasts, respectively, and elucidates the correlation structure of the gene expression at various time points with each cell type present. (A) Plot of PCA scores, t₁ and t₂, separating the observations by two PCs that explain 51.1% and 17.8% of the variance in data, respectively. Dashed line represents the 95% confidence limit of the distribution of scores. (B) Plot of PCA loadings, p₁ and p₂, that shows the correlation of the gene expression data with the sources of maximum variance. Model quality parameters: R²X=0.689, Q²=0.450. (C) Plot of PLS-DA scores, t₁ and t₂, for observations (cell type and culture type) that segregate three distinct cell types by two LVs. (D) Loading plot depicting the correlation structure of the gene expression data and the corresponding cell types, indicating (1) the weights, w*, that combine the X-variables (gene expression values at different time points) to form the scores, t; and (2) the weights, c, of the discriminating Y-variables (corresponding to each cell type). Gene expression values at specific times (X-variables, triangles) that contribute most to the cell type classification (Y-variables, circles) are labeled accordingly with the corresponding color scheme. R²Y=0.750, Q²=0.681. PCs, principal components; PCA, principal component analysis; PLS-DA, partial least squares discriminant analysis; LVs, latent variables. Color images available online at www.liebertonline.com/tea

FIG. 6.

PLS-DA models of single cell types can robustly separate cell samples derived from different culture conditions and describe the important gene expression variables that correlate with each response to co- or tri-culture. (A, D) Model discriminating between adipocytes from co- and tri-cultures; R²Y=0.898, Q²=0.820. (B, E) Model discriminating between osteoblasts from co- and tri-cultures; R²Y=0.972, Q²=0.920. (C, F) Model discriminating between MSCs from different co- and tri-culture conditions; R²Y=0.854, Q²=0.716. (A–C) Score plots of clusters of adipocytes (A), osteoblasts (B), and MSCs (C) segregated into distinct groups by one (adipocytes or osteoblasts) or two (MSCs) latent variables. Dashed lines represent the 95% confidence limit of the distribution of scores for the corresponding model. (D–F) Loading plots depicting the correlation structure of gene expression data and the corresponding observation sets for each model. Gene expression values at specific times (X-variables) that significantly contribute (p<0.05) most to the cell type classification (Y-variables) are shaded accordingly with the corresponding color scheme. Bar graphs depict mean±standard error of the mean (S.E.M). Color images available online at www.liebertonline.com/tea