高效诱导人角质细胞干细胞分化为功能性成釉细胞。
Efficient induction of functional ameloblasts from human keratinocyte stem cells.
机构信息
Southern Center for Biomedical Research, Fujian Normal University, Fuzhou, 350108, China.
Fujian Key Laboratory of Developmental and Neural Biology, College of Life Science, Fujian Normal University, Fuzhou, 350108, China.
出版信息
Stem Cell Res Ther. 2018 May 2;9(1):126. doi: 10.1186/s13287-018-0822-4.
BACKGROUND
Although adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.
METHODS
Cultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.
RESULTS
Treatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.
CONCLUSIONS
Application of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human-mouse chimeric tooth germs.
背景
虽然成人组织来源的表皮干细胞与具有成牙潜能的小鼠牙间质重组成嵌合体后，在体外可分化为分泌釉质的成釉细胞，并形成具有再生釉质的牙，但诱导率相对较低。此外，再生釉质是否保留与人类釉质相同的棱柱运行模式并获得可与人类釉质相媲美的机械性能，确实需要进一步研究。
方法
用成纤维细胞生长因子 8（FGF8）和 Sonic hedgehog（SHH）处理培养的人角质形成细胞干细胞（hKSCs）18 小时或 36 小时，然后将其与 E13.5 小鼠牙间质重组成嵌合体牙胚，并将 FGF8 和 SHH 浸泡的琼脂糖珠植入牙胚中。将重组牙胚植入裸鼠肾囊中进行培养。在不同时间点采集样本，进行组织学、免疫组织化学、TUNEL 和 Western blot 分析。扫描电子显微镜和纳米压痕试验进一步分析了再生釉质的棱柱运行模式和机械性能。
结果
在组织重组合之前，用 FGF8 和 SHH 处理 hKSCs 可将牙样结构形成率提高到约 70%。FGF8 和 SHH 显著增强了培养的 hKSCs 的干细胞特性。扫描电子显微镜分析显示，再生釉质的棱柱运行模式与人类釉质相似。纳米压痕试验表明，尽管再生釉质的机械性能远低于人类儿童和成年小鼠的釉质，但随着培养时间的延长，机械性能有所提高。
结论
FGF8 和 SHH 蛋白在培养的 hKSCs 中的应用可提高其干细胞特性，但不能促进 hKSCs 的成牙命运，从而提高 hKSCs 的成釉细胞分化效率和人-鼠嵌合体牙胚的牙齿形成效率。
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