Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells
Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells
复制标题
DOI：
10.1177/0022034515611602
复制
发表时间：
2016-01-01
影响因子：
7.6
通讯作者：
Egusa, H.
中科院分区：
文献类型：
--
作者：
Yu, G.;Okawa, H.;Egusa, H.
人类牙龈成纤维细胞（hGF）是诱导多能干细胞（iPSC）的一个有吸引力的来源，有望成为再生牙科的有力工具。然而，在临床应用之前需要解决的问题包括使用动物源性饲养细胞进行培养。本研究的目的是通过评估hGFs的饲养能力来建立自体hGF-derived iPSC（hGF-iPSC）培养系统。在含血清和无血清培养基中，hGFs显示出比人真皮成纤维细胞（hDFs）更高的增殖。在无血清条件下分离出三种HGF菌株，尽管2种显示增殖受损。当将hGF-iPSC转移到丝裂霉素C灭活的hGF、hDF或小鼠来源的SNL饲养细胞上时，HGF和SNL饲养细胞明显支持hGF-iPSC超过50代，而HDF饲养细胞仅能够维持未分化的hGF-iPSC生长几代。在HGF饲养细胞上传代20次后，在HGF和SNL饲养细胞上培养的hGF-iPSC的NANOG和OCT 3/4启动子处的胚胎干细胞标志物表达和CpG甲基化相似。hGF-iPSC在HGF饲养细胞上的长期培养维持其正常核型和多能性。在HGF饲养层上，hGF-iPSC集落被许多集落来源的成纤维细胞样细胞包围，传代7 d后完整集落的大小明显大于SNL饲养层。同种异体HGF菌株也维持hGF-iPSC 10代。与hDF相比，hGF显示出更高的层粘连蛋白-332、层粘连蛋白α 5链和胰岛素样生长因子-II的产生，据报道这些物质维持多能干细胞的长期自我更新。这些结果表明，hGF具有优异的饲养能力，因此可以用作常规小鼠来源的SNL和HDF饲养器的替代品。此外，我们的研究结果表明，HGF饲养细胞是自体hGF-iPSCs的无动物成分离体扩增的有希望的候选者，从而为hGF-iPSCs的未来治疗应用提供了重要的一步。
Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin alpha 5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs.