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 triculture 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.

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

“…25 In the experiments presented here, cell viability was generally maintained for the duration of the experiment in all cell types (Fig. 3, 5, and 7B).…”

“…1). 25 An opaque photomask was used in subsequent steps to prevent any further UV light exposure and crosslinking. Each subsequent layer is laminated to the prior and constructs do not separate over the course of the experiment or experience changes in tensile properties, as demonstrated in prior work.…”

“…Each subsequent layer is laminated to the prior and constructs do not separate over the course of the experiment or experience changes in tensile properties, as demonstrated in prior work. 25,26,44 Laminated constructs were extracted from the device and sectioned with a scalpel perpendicular to the long axis of the laminate to yield up to eighteen 1.5 mm-wide mono-, co-, and tri-culture constructs (Fig. 1).…”

“…However, in many of these systems, both separation of unique cell populations for analysis and retrieval of live cells for further experimentation are difficult. Therefore, this study demonstrates the development and implementation of a 3D tri-culture platform that permits individual cell type analysis 25 and on-demand retrieval and further analysis of live MSCs post-culture. To achieve live cell retrieval, a matrix metalloprotease (MMP)-degradable poly(ethylene-glycol) (PEG)-based material was included in the culture system.…”

Opportunities and challenges in three-dimensional brown adipogenesis of stem cells

“…Adipose tissue in vivo is composed of multiple cell types, such as preadipocytes, differentiated adipocytes, stem cells, adipose stromal cells/fibroblasts, endothelial vascular cells, and perivascular support cells, embedded within an extensive 3D ECM network. Therefore, 3D multi-cellular culture has the potential to more efficiently recapitulate the complex stem cell and/or adipose tissue microenvironment to mimic the physiology and pathology of adipocytes (Hammoudi et al, 2012; Rinker et al, 2014; Sorrell et al, 2011). …”