Three-Dimensional In Vitro Tri-Culture Platform to Investigate Effects of Crosstalk Between Mesenchymal Stem Cells, Osteoblasts, and Adipocytes

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.