Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.
Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.
复制标题
新型细胞培养模式可延长小鼠角膜上皮细胞的体外和体内增殖活性
DOI：
10.3389/fcell.2021.675998
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发表时间：
2021
影响因子：
5.5
通讯作者：
Li C
中科院分区：
文献类型：
--
作者：
An X;Wang G;Jin M;Zhou X;Gao S;Chen J;Reinach PS;Liu Z;Xue Y;Li C
从细胞培养中获得足够量的小鼠角膜上皮细胞（mCEC）进行移植手术一直是一个长期的挑战。这种限制可归因于其增殖活性的传代依赖性下降。我们在这里描述了一种新的6C培养基，其中含有六种不同的调节剂不同的信号通路，控制增殖的mCEC活性的发展。它的使用缩短了获得上皮片所需的时间和精力，以加速实验动物模型中上皮伤口的愈合。该无血清6C培养基含有：Y27632、毛喉素、SB 431542、DAPT、IWP-2、LDN-193189以及DermaLife K角质形成细胞钙。它们的加入抑制了上皮间充质转分化的四种特异性标志物的增加：ZEB 1/2、Snail、β-连环蛋白和α-SMA。将该培养基应用于无饲养层的气升系统中以获得足够的上皮祖细胞群，由于上皮祖细胞转分化为上皮间充质细胞的抑制，促进了上皮祖细胞的获取。基于P63、K14、Pax 6和K12基因表达水平的不变性，证实了转分化下降的减少。这种细胞培养技术有望促进细胞命运决定机制的离体表征。此外，其实施将提高祖小鼠角膜上皮细胞的产量，这增加了使用这些细胞作为来源以产生上皮片的可能性，所述上皮片用于进行移植手术以在临床环境中治疗角膜缘干细胞缺陷。此外，从这些研究中获得的新见解有望改善角膜再生医学的结果。
It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.