Sidney Laura E, McIntosh Owen D, Hopkinson Andrew
Invest Ophthalmol Vis Sci. 2015 Nov;56(12):7225-35. doi: 10.1167/iovs.15-17810.
Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC.
Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin), and MSC markers (CD73, CD90, and CD105) were compared in CEC and CSC by immunocytochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton's jelly (WJ-MSC).
In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12, and CK19, but not other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC expressed lower levels of CK3, CK12, and CK19, but also stained for pan cytokeratin and expressed KRT5.
Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way in which differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognized.
角膜上皮细胞和基质细胞在体内可通过不同的中间丝加以区分，角膜上皮细胞（CEC）表达细胞角蛋白，角膜细胞表达波形蛋白。分离培养的角膜细胞会发生表型改变，失去角膜细胞标志物的表达，并获得与间充质基质细胞（MSC）相关的标志物。本研究调查了与CEC相比，培养的角膜基质细胞（CSC）中间丝表达相关的这种表型变化。
通过免疫细胞化学和逆转录定量聚合酶链反应（RT-qPCR）比较CEC和CSC中上皮标志物（CK3、CK12、CK19、泛细胞角蛋白、E-钙黏蛋白）、角膜细胞标志物（CD34、波形蛋白）和MSC标志物（CD73、CD90和CD105）的表达。在CSC培养的不同阶段评估表达情况，并与从脐带华通氏胶中提取的另一种基质细胞类型（WJ-MSC）进行比较。
体内角膜细胞不表达细胞角蛋白。然而，培养的CSC表达与上皮相关的CK3、CK12和CK19，但不表达其他细胞角蛋白。随着CSC传代，细胞角蛋白的表达增加，而当CSC诱导进入静止状态时表达降低。相比之下，WJ-MSC表达较低水平的CK3、CK12和CK19，但也有泛细胞角蛋白染色并表达KRT5。
培养的CSC在培养过程中发生表型变化，表达通常与CEC相关的特定细胞角蛋白丝。细胞角蛋白表达始于细胞在塑料上培养时，并随着传代而增加。这一发现可能会影响区分培养的CEC和CSC的方式。研究人员需要意识到，细胞角蛋白的表达不一定代表上皮污染，并且CEC和CSC可能比之前认为的更具相关性。
