Establishment of Periodontal Ligament Stem Cell-like Cells Derived from Feeder-Free Cultured Induced Pluripotent Stem...

The periodontal ligament (PDL) is a fibrous connective tissue that connects the cementum of the root to the alveolar bone. PDL stem cells (PDLSCs) contained in the PDL can differentiate into cementoblasts, osteoblasts, and PDL fibroblasts, with essential roles in periodontal tissue regeneration. Therefore, PDLSCs are expected to be useful in periodontal tissue regeneration therapy. In a previous study, we differentiated induced pluripotent stem cells (iPSCs) into PDLSC-like cells (iPDLSCs), which expressed PDL-related markers and mesenchymal stem cell (MSC) markers; they also exhibited high proliferation and multipotency. However, the iPSCs used in this differentiation method were cultured on mouse embryonic fibroblasts; thus, they constituted on-feeder iPSCs (OF-iPSCs). Considering the risk of contamination with feeder cell-derived components, iPDLSCs differentiated from OF-iPSCs (ie, OF-iPDLSCs) are unsuitable for clinical applications. In this study, we aimed to obtain PDLSC-like cells from feeder-free iPSCs (FF-iPSCs) using OF-iPDLSC differentiation method. First, we differentiated FF-iPSCs into neural crest cell-like cells (FF-iNCCs) and confirmed that FF-iNCCs expressed NCC markers (eg, Nestin and p75NTR). Then, we cultured FF-iNCCs on human primary PDL cell-derived extracellular matrix for 2 weeks; the resulting cells were named FF-iPDLSCs. FF-iPDLSCs exhibited higher expression of PDL-related and MSC markers compared with OF-iPDLSCs. FF-iPDLSCs also demonstrated proliferation and multipotency in vitro. Finally, we analyzed the ability of FF-iPDLSCs to form periodontal tissue in vivo upon subcutaneous transplantation with β-tricalcium phosphate scaffolds into dorsal tissues of immunodeficient mice. Eight weeks after transplantation, FF-iPDLSCs had formed osteocalcin-positive bone/cementum-like tissues and collagen 1-positive PDL-like fibers. These results suggested that we successfully obtained PDLSC-like cells from FF-iPSCs. Our findings will contribute to the development of novel periodontal regeneration therapies.