Abstract：
Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.
摘要：
使用人诱导多能干细胞（hiPSCs）进行牙齿再生的研究对于自体牙齿再生很有价值。获得间充质和上皮细胞作为牙齿再生的资源是必要的，因为间充质-上皮相互作用在牙齿发育中起着至关重要的作用。我们报道了hiPSCs来源的牙齿上皮样细胞（EPI-iPSCs）的建立，但hiPSCs来源的牙体间充质干细胞(MSCs)尚未见报道。本研究旨在建立hiPSCs来源的MSCs，并用EPI-iPSC将它们分化为牙齿细胞。考虑到牙科骨髓间充质干细胞来源于神经嵴，通过神经c形成诱导hiPSCs分化为MSCs，以获得牙科MSCs的特性。通过神经c形成将hiPSCs分化为MSCs，建立的hiPSCs与PA6基质细胞培养和分化，分化的hiPSCs在超低附着板上形成神经球。在补充血清的培养基中将神经球分化成MSC。神经c介导的MSC（NC-MSC）连续显示典型的MSC形态和表达的MSC标记。牙源性诱导8天后，在不与牙上皮细胞共培养的情况下,NC-MSCs单独组的牙源性/矿化相关基因和牙本质硅磷蛋白(DSPP)蛋白表达水平升高.NC-MSCs和EPI-iPSCs共培养组显示出高表达水平的牙釉质生成/牙质生成/矿化相关基因和DSPP蛋白。此外，NC-MSC和EPI-iPSC共培养组比单独的NC-MSC组早产生钙沉积。这些结果表明,来自hiPSC的建立的NC-MSC具有与牙齿上皮细胞的牙齿分化能力。此外,证实了hiPSCs来源的牙科干细胞可能是一种用于自体牙齿再生的新型细胞来源。
