Periodontal ligament stem cells (PDLSCs) play central roles in periodontal ligament (PDL) tissue homeostasis, repair, and regeneration. Previously, we established a protocol to differentiate human-induced pluripotent stem cell-derived neural crest-like cells (iNCs) into PDLSC-like cells (iPDLSCs) using human PDL cell-derived extracellular matrix (ECM). However, it remained unclear what factors principally regulate the differentiation of iNCs into iPDLSCs. In this study, we aimed to identify the transcription factor regulating production of human PDL cell-derived ECM, which is responsible for the generation of iPDLSCs. We cultured iNCs on ECMs of two human PDL cell lines (HPDLC-3S and HPDLC-3U) and of human dermal fibroblasts (HDF). iNCs cultured on HPDLC-3U demonstrated higher iPDLSC-associated gene expression and mesenchymal differentiation capacity than cells cultured on HDF or HPDLC-3S. The transcription factor PAX9 was highly expressed in HPDLC-3U compared with HDF and HPDLC-3S. iNCs cultured on siPAX9-transfected HPDLC-3U displayed downregulation of iPDLSC-associated marker expression and adipocytic differentiation capacity relative to controls. Our findings suggest that PAX9 is one of the transcription factors regulating ECM production in human PDL cells, which is responsible for the differentiation of iNCs into iPDLSCs.

Activin A, a member of transforming growth factor-β superfamily, is involved in the regulation of cellular differentiation and promotes tissue healing. Previously, we reported that expression of activin A was upregulated around the damaged periodontal tissue including periodontal ligament (PDL) tissue and alveolar bone, and activin A promoted PDL-related gene expression of human PDL cells (HPDLCs). However, little is known about the biological function of activin A in alveolar bone. Thus, this study analyzed activin A-induced biological functions in preosteoblasts (Saos2 cells). Activin A promoted osteoblastic differentiation of Saos2 cells. Activin receptor-like kinase (ALK) 1, an activin type I receptor, was more strongly expressed in Saos2 cells than in HPDLCs, and knockdown of ALK1 inhibited activin A-induced osteoblastic differentiation of Saos2 cells. Expression of ALK1 was upregulated in alveolar bone around damaged periodontal tissue when compared with a nondamaged site. Furthermore, activin A promoted phosphorylation of Smad1/5/9 during osteoblastic differentiation of Saos2 cells and knockdown of ALK1 inhibited activin A-induced phosphorylation of Smad1/5/9 in Saos2 cells. Collectively, these findings suggest that activin A promotes osteoblastic differentiation of preosteoblasts through the ALK1-Smad1/5/9 pathway and could be used as a therapeutic product for the healing of alveolar bone as well as PDL tissue.

Periodontal ligament stem cells (PDLSCs) play central roles in periodontal ligament (PDL) tissue homeostasis, repair, and regeneration. Previously, we established a protocol to differentiate human induced pluripotent stem cell-derived neural crest-like cells (iNCs) into PDLSC-like cells (iPDLSCs) using human PDL cell-derived extracellular matrix (ECM). However it remained unclear what factors principally regulate the differentiation of iNCs into iPDLSCs. In this study, we aimed to identify the transcription factor regulating production of human PDL cell-derived ECM, which is responsible for the generation of iPDLSCs. We cultured iNCs on two human PDL cell lines (HPDLC-3S and HPDLC-3U) and human dermal fibroblasts (HDF). iNCs cultured on HPDLC-3U showed higher iPDLSC-associated gene expression and mesenchymal differentiation capacity than cells cultured on HDF or HPDLC-3S. The transcription factor PAX9 was highly expressed in HPDLC-3U compared with HDF and HPDLC-3S. iNCs cultured on siPAX9-transfected HPDLC-3U displayed downregulation of iPDLSC-associated marker expression and adipocytic differentiation capacity relative to controls. Our findings suggest that PAX9 is one of the transcription factors regulating ECM production in human PDL cells, which is responsible for the differentiation of iNCs into iPDLSCs.

The aim of this study is to clarify the biological functions of decorin (DCN) in the healing and regeneration of wounded periodontal tissue. We investigated the expression pattern of DCN during the healing of wounded periodontal tissue in rats by immunohistochemistry and the effects of DCN on the osteoblastic differentiation of human periodontal ligament (PDL) stem cells (HPDLSCs) and preosteoblasts by Alizarin red S staining, quantitative reverse transcription-polymerase chain reactions, and western blotting. The expression of DCN was increased around the wounded PDL tissue on day 5 after surgery compared with the nonwounded PDL tissue, whereas its expression was not changed in the osteoblastic layer around the wounded alveolar bone. Furthermore, DCN promoted the osteoblastic differentiation of HPDLSCs, but it did not affect the osteoblastic differentiation of preosteoblasts. ERK1/2 phosphorylation was upregulated during the DCN-induced osteoblastic differentiation of HPDLSCs. DCN did not affect proliferation, migration, or the PDL-related gene expression of HPDLSCs. In conclusion, this study demonstrates that DCN has a role in the healing of wounded periodontal tissue. Furthermore, DCN secreted from PDL cells may contribute to bone healing by upregulating osteoblastic differentiation through ERK1/2 signaling in HPDLSCs, indicating a therapeutic effect of DCN in periodontal tissue regeneration.