Characterization Of Co-Culture Of Oral Mucosa Spheroids And MSCs

Spontaneous spheroids culture is a novel three-dimensional culture strategy for the rapid and efficient selection of progenitor cells. In this study, we established the co-culture of oral mucosa spheroids and mesenchymal stem cells (MSCs), and investigated the periodontal tissue regeneration capacity. Primary cells were isolated from the oral mucosa and bone marrow of C57BL/6J mice. The mucosa cells were seeded into low-adherence specific culture plates to form spontaneous spheroids. The stemness and pluripotency of spheroids were investigated by RT-qPCR and immunofluorescence. After spheroids and MSCs co-cultured, and multi-differentiation capacities (osteo-, cementum-, and ligament-differentiations) were investigated. Oral mucosa cells successfully formed spontaneous spheroids after 24 h. spheroids were positive for pluripotency markers (Oct4, Sox2, SSEA1) and neural crest makers (Nestin, βⅢ-Tubulin). After co-culture of spheroids and MSCs, the cells were successfully differentiated into osteoblasts, cementoblasts and ligament-related cells under the corresponding differentiation conditions. Higher expression of osteogenic markers, cementoblast markers (CEMP1, DMP1), and ligament-related markers (Tnmd, Scleraxis) was found in co-cultured cells. Spontaneous spheroids from oral mucosa enhance stemness and pluripotency. The co-culture of spheroids and MSCs showed superior periodontal tissue regeneration ability, which may be a potent therapy for oral and craniomaxillofacial tissue regeneration.