Differentiation of iPS Cells into Periodontal Ligament Cells

Periodontal ligament (PDL) is a thin connective tissue that connects the tooth to the bony socket and plays a crucial role in the regeneration and maintenance of homeostasis of periodontal tissues by supplying stem/progenitor cells. Induced pluripotent stem cells (iPSCs) are highly anticipated in regenerative medicine because of their differentiation potential into a wide variety of cell types. In this study, we investigated the effects of humoral factors on iPSC differentiation by culturing iPSCs in the presence of PDL cell-derived culture supernatants. Changes in gene expression were analyzed using quantitative real-time PCR, reverse-transcription PCR, and RNA sequencing. The marker protein expression on the cell surface was assessed using flow cytometry. Periodontal regeneration was verified by microcomputed tomography and histomorphological observation in a periodontal defect model using male F344/NJcl-<i>rnu</i>/<i>rnu</i> rats. When iPSCs were cultured in the PDL culture supernatant, some cells formed clumps, and spindle-shaped cells grew out from them. Upon passaging, spindle cells increased further, and by the fifth passage, these cells occupied the entire culture. These cells (iPS-PDLs) expressed genes such as periostin and Asporin/PLAP1, and their comprehensive gene expression patterns resembled those of PDL cells. iPS-PDL cells exhibited a cell surface antigen profile of CD90+, CD73+, CD105+, CD44+, CD29+, CD14-, CD34-, CD45-, and CD19- and differentiation potential into osteoblasts, adipocytes, and chondrocytes. Transplantation of iPS-PDLs into rat periodontal defects increased the height of newly formed bone and enhanced periodontal tissue regeneration after 4 weeks. Our results showed that iPSCs differentiated into cells with properties similar to those of PDL cells in the presence of humoral factors of cultured PDL cells. Additionally, the transplantation of iPS-PDL cells into periodontal defects induces periodontal tissue regeneration. These findings provide valuable insights for developing novel periodontal regenerative therapies using iPSCs.