Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na⁺ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na⁺ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K⁺ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K⁺ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K⁺ uptake and severe K⁺ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.