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PAX9 regulates periodontal ligament stem cell-like differentiation of human induced pluripotent stem cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208250 | GEO | 2022/07/18

REPOSITORIES: GEO

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Project description:The pre-occlusal eruption brings the rat molars into functional occlusion, which implicates tensional strains during mastication. We hypothesized that upon establishment of occlusion, the periodontal ligament undergoes cell and extracellular matrix maturation to adopt to this mechanical function. We thus aimed to characterize the protein content and changes in expression levels of tensionally relevant extracellular matrix components in the periodontal ligament between the eruption and newly established occlusion in rat molars. Twelve Wistar male rats were divided into 3 groups based on eruption and occlusal stages: covering the pre-occlusal stages at time of eruption, the first day of occlusion and 1 week after occlusion. We employed laser capture microdissection to obtain samples of the periodontal ligament at three regions, cervical, apical and subapical. The proteome was screened with Tandem Mass Tag 10-plexTM mass spectrometry and the expression of key proteins were confirmed by immunofluorescence. Differential expression of matrisome proteins was seen in the cervical and the apical region. Downregulation of pro-metabolic proteins, such as apolipoproteins implicated in lipid transport was observed. Alpha-fetoprotein, a stem cell marker, was also downregulated indicating cell differentiation and PDL maturation. Upregulated proteins were components of the extracellular matrix, involving several proteoglycans and glycoproteins and the matrisome was thus further analyzed. Periostin appeared around collagen α-1 (III) fibers and its expression was particularly strong on Sharpey’s fibers. This co-localization coincided with organization of collagen fibers in direction of the occlusal forces. Establishment of occlusion coincides with cellular differentiation and the maturation of the extracellular matrix of the periodontal ligament, as seen by downregulation of the stem cell marker Alpha-fetoprotein and apolipoproteins, and the progressive accumulation of collagen type III fibers, proteoglycans, glycoproteins. The increase in collagen fiber associates periostin may reflect a physiological response to enforce cell-ECM contacts during PDL maturation.

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PAX9 regulates periodontal ligament stem cell-like differentiation of human induced pluripotent stem cells

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