Agarose-based spheroid culture enhanced stemness and promoted odontogenic differentiation potential of human dental follicle cells in vitro

Human dental follicle cells (HDFCs) are an ideal cell source of stem cells for dental tissue repair and regeneration and they have great potential for regenerative medicine applications. However, the conventional monolayer culture usually reduces cell proliferation and differentiation potential due to the continuous passage during in vitro expansion. In this study, primary HDFC spheroids were generated on 1% agarose, and the HDFCs spontaneously formed cell spheroids in the agarose-coated dishes. Compared with monolayer culture, the spheroid-derived HDFCs exhibited increased proliferative ability for later passage HDFCs as analysed by Cell Counting Kit-8 (CCK-8). The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the expression of stemness marker genes Sox2, Oct4 and Nanog was increased significantly in the HDFC spheroids. Furthermore, we found that the odontogenic differentiation capability of HDFCs was significantly improved by spheroid culture in the agarose-coated dishes. On the other hand, the osteogenic differentiation capability was weakened compared with monolayer culture. Our results suggest that spheroid formation of HDFCs in agarose-coated dishes partially restores the proliferative ability of HDFCs at later passages, enhances their stemness and improves odontogenic differentiation capability in vitro. Therefore, spheroid formation of HDFCs has great therapeutic potential for stem cell clinical therapy.