Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration
Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration
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DOI：
10.1089/scd.2018.0021
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发表时间：
2018-04-23
影响因子：
4
通讯作者：
Dilley, Rodney J.
中科院分区：
文献类型：
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作者：
Liew, Lawrence J.;Chen, Linda Q.;Dilley, Rodney J.
鼓膜 (TM) 中已鉴定出具有干细胞样特征的表皮细胞，并且专门定位于鼓膜和环状区域。虽然它们被认为在急性和慢性 TM 穿孔的再生中发挥作用，但其作用机制和调节的证据尚未描述。事实上，这些假定的干细胞的行为在很大程度上是未知的，部分原因是缺乏有效细胞分离的精细方法。在这项研究中，我们使用正常和穿孔的大鼠 TM 组织比较了不同的外植体技术，并研究了它们的离体特征。体内穿孔后的 TM 显示表皮干细胞标记物整合素 1 和细胞角蛋白 (CK) 19 以及增殖 Ki-67 的染色增加，表明增殖中心的激活。从外植体培养物中分离出成纤维细胞和上皮细胞的混合群。将切除的 TM umbo 植入培养孔插入物上是最有效的技术。由穿孔TM制成的外植体先于由未穿孔TM制成的外植体产生细胞。更重要的是，植入的 TM umbo 类器官能够以连续的方式产生细胞，从而允许随后使用胰蛋白酶进行收获。全细胞角蛋白阳性的原代大鼠TM上皮细胞培养物具有集落形成活性，并且可以富集CK 19阳性细胞，这些细胞在进行伤口测定时能够通过增殖和细胞迁移进行培养物扩增。总而言之，TM 创伤激活了增殖中心，并促进了 TM umbo 类器官培养物的早期细胞产生，产生了 TM 干细胞样培养物，被证明适合 TM 的组织工程。
Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin 1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM.