Human periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration. The generation of PDLSCs from human induced pluripotent stem cells (iPSCs) by simulating the development pattern of PDLSCs in vivo provided a new way to obtain a large and stable source of PDLSCs. However, animal-derived components were still necessary for current differentiation protocols, which could cause safety and ethical problems and hinder the clinical application of iPSCs-derived PDLSCs. Here, we established a novel protocol to induce iPSCs into PDLSCs by chemically defined conditions. We first induced iPSCs into neural crest-like cells by inhibiting TGF-β pathway, BMP pathway and Notch pathway using SB431542, LDN and DAPT, respectively. The iPSC-induced neural crest-like cells were further cultured in chemically defined medium containing recombinant human bFGF as well as the rho-associated protein kinase inhibitor Y27632 to generate PDLSCs. The characteristics of iPSCs-derived PDLSCs and the bi-potentiality of osteogenesis and adipogenesis differentiation were verified in vitro. The establishment of the chemically defined differentiation system breaks through the limitation brought from animal-derived components and enables us to obtain a large number of PDLSCs, which holds a significant value to the research and treatment of periodontal diseases.

人牙周膜干细胞（PDLSCs）在牙周组织再生中发挥着重要作用。通过模拟人诱导多能干细胞（iPSCs）在体内的发育模式生成PDLSCs，为获得大量稳定的PDLSCs来源提供了新的途径。然而，目前的分化方案仍然需要动物源性成分，这可能会导致安全和伦理问题，并阻碍 iPSCs 衍生的 PDLSCs 的临床应用。在这里，我们建立了一种新的协议，通过化学定义的条件将 iPSCs 诱导为 PDLSCs。我们首先分别使用 SB431542、LDN 和 DAPT 通过抑制 TGF-β 通路、BMP 通路和 Notch 通路将 iPSCs 诱导为神经嵴样细胞。iPSC 诱导的神经嵴样细胞在含有重组人 bFGF 和 rho 相关蛋白激酶抑制剂 Y27632 的化学成分确定的培养基中进一步培养，以产生 PDLSC。在体外验证了 iPSCs 衍生的 PDLSCs 的特征以及成骨和脂肪形成分化的双潜能。化学定义分化系统的建立突破了动物源性成分带来的限制，使我们能够获得大量的PDLSCs，这对牙周病的研究和治疗具有重要价值。在体外验证了 iPSCs 衍生的 PDLSCs 的特征以及成骨和脂肪形成分化的双潜能。化学定义分化系统的建立突破了动物源性成分带来的限制，使我们能够获得大量的PDLSCs，这对牙周病的研究和治疗具有重要价值。在体外验证了 iPSCs 衍生的 PDLSCs 的特征以及成骨和脂肪形成分化的双潜能。化学定义分化系统的建立突破了动物源性成分带来的限制，使我们能够获得大量的PDLSCs，这对牙周病的研究和治疗具有重要价值。