Cell-fate decision of mesenchymal stem cells toward osteocyte differentiation is committed by cell condensed condition in spheroid culture

Osteocytes are mechanosensory commander cells to regulate bone remodeling throughout the lifespan. While the osteocyte is known as a terminally differentiated cell derived from mesenchymal stem cell, the detailed mechanisms of cell-fate decision toward osteocyte differentiation still remain unclear. In this study, we fabricated three-dimensional (3D) self-organized spheroids using human mesenchymal stem cells (hMSCs). Under the osteogenesis induction medium, the spheroid culture model exerted the osteocyte-likeness within 2 days compared to a conventional 2D monolayer model. By using an inhibitor of actin polymerization, we showed an involvement of actin balancing in the osteocyte differentiation in the spheroid. Notably, we represented that the cell condensed condition acquired in the 3D spheroid culture model determined a differentiation fate of MSCs to osteocytes via actin balancing. Taken together, we suggest that our self-organized spheroid model can be utilized as a new in vitro model to represent the osteocyte differentiation process and further to recapitulate an in vitro ossification process.