Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.