Spheroid size-induced apoptosis enhances osteogenic differentiation of iPS cells

Induced pluripotent stem cells (iPSCs) have significant potential for regenerative medicine, particularly for bone tissue engineering. While three-dimensional spheroid cultures enhance iPSC differentiation by better mimicking physiological conditions, spheroid size critically affects cell viability and differentiation ability. Microwell plates enable large-scale production of uniform spheroids and would be especially useful for regenerative medicine and tissue engineering. Here, we investigated the effect of spheroid size on the osteogenic differentiation of iPSCs using microwell plates to generate spheroids under the following conditions: Elp200 (microwell plate with 200/100 μm diameter/depth) and Elp900 (microwell plate with 900/700 μm). We observed that larger Elp900 spheroids promoted mesodermal differentiation more effectively, likely due to enhanced cell-cell interactions and altered internal microenvironments. However, Elp900 spheroids exhibited increased apoptosis in their core regions, evidenced by viability staining, transmission electron microscopy, and TUNEL staining. Upon dissociation and adherent culture, Elp900-derived cells demonstrated significantly higher expression of osteogenic markers (Runx2, Ibsp) and mineralization compared to Elp200-derived cells. Proteomic analysis revealed that apoptosis- and extracellular matrix (ECM)-related proteins, such as SERPINH1 and COL4A1, were upregulated in Elp900 cultures. These findings suggest that controlled apoptosis within larger spheroids may activate stress-related pathways, promote ECM formation, and enhance osteogenic differentiation by activating the TGF-β signaling pathway. Our findings highlight optimal spheroid sizing as a key factor for maximizing the efficiency and reproducibility of osteogenic differentiation of iPSCs, providing a foundation for improved strategies in iPSC-based bone tissue regeneration.