Cell death, cavitation and spontaneous multi-differentiation of dental pulp stem cells-derived spheroids in vitro: A journey to survival and organogenesis

Background information. During embryonic development, cell death transforms the solid embryonic cell mass into a hollow structure (cavitation), which allows the surviving cells to differentiate into varied tissues and organs around the cavity. This process can be partly reproduced with embryonic stem cells. However, it is unknown if adult stem cell masses have the same ability to cavitate and then differentiate into organs. In this study, we assessed the capacity of human dental pulp stem cells (DPSCs)-derived spheroids to mimic the above-mentioned cavitation and spontaneous differentiation in vitro. Results. DPSCs were able to form large-sized spheroids on matrigel in osteogenic medium. Inside the spheroids, cells in the centre showed positive stain to stem cell markers, alkaline phosphatase and STRO-1. Hypoxia and massive cell death were observed in the core of the spheroids. Cavities were formed when the spheroids were cultivated in the osteogenic medium for about 14 days. After 28 days of cultivation, the surviving cells around the cavity spontaneously differentiated into neuronal (28.8%), vascular (33.3%), osteogenic (46.7%) and cartilaginous (72.0%) tissues under the osteogenic medium only. In contrast, when DPSCs-formed cell sheets were folded into giant-sized lumps and cultivated under the same conditions, the folded cell sheets became an entire lumenal structure and failed to differentiate into neuronal, osteogenic and cartilaginous cells. Marker analysis showed that cavitation-related molecules BMP7 and FGF3 expressed on the wall of the cavity in the spheroids, suggesting that the cavitation was functional, whereas cavitation-related molecules were absent in the folded cell sheets. Conclusions. DPSC-derived spheroids can mimic the developmental process of cell survival, cavitation and spontaneous multi-differentiation on matrigel under certain conditions. This work allows for functional studies to investigate organ regeneration with human DPSCs in vitro.