Liu Qing, Guo Yonglong, Liu Shiwei, Wang Peiyuan, Xue Yunxia, Cui Zekai, Chen Jiansu
Ophthalmology Department, The People's Hospital of Yubei District of Chongqing city, Chongqing, China.
Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China.
PeerJ. 2019 Apr 16;7:e6734. doi: 10.7717/peerj.6734. eCollection 2019.
Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na-K-ATPase. The gene expression levels of NIFK, Na-K-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.
角膜内皮细胞（CECs）维持角膜透明度和视力。然而，这些细胞在体外的增殖能力有限，这促使研究人员寻找有效的培养技术。我们研究的目的是评估从诱导多能干细胞（iPSCs）获得的条件培养基（CM）作为牛角膜内皮细胞（B - CECs）有效增殖来源的用途。在我们的研究中，当细胞在25%的iPSC条件培养基（iPSC - CM）中生长时，B - CECs的增殖能力适度增强。此外，六边形细胞形态维持到第4代，这与对照角膜内皮培养基（CEM）中观察到的不规则和增大的形状相反。25% iPSC - CM组和CEM组的B - CECs均表达Na - K - ATP酶。iPSC - CM组中NIFK、Na - K - ATP酶、Col4A和Col8A的基因表达水平以及进入S期和G2期的细胞百分比更高。iPSC - CM组中的凋亡细胞数量也减少。与对照培养相比，iPSC - CM促进细胞迁移，并且这些细胞在传代几次后表现出更好的屏障功能。还研究了iPSC - CM介导的细胞增殖机制，在B - CECs暴露于iPSC - CM后观察到Akt的磷酸化，并且在iPSC - CM中显示持续磷酸化长达180分钟。我们的研究结果表明，iPSC - CM可能利用PI3 - 激酶信号传导来调节细胞周期进程，这可导致细胞增殖增强。CM的有效成分分析表明，在iPSC - CM组中，激活素 - A的表达显著增加。如果添加激活素 - A作为补充剂，它可以帮助维持细胞形态，类似于CM。因此，我们得出结论，激活素 - A是CM中促进细胞增殖和维持细胞形态的有效成分之一。
