Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco’s modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

中文翻译：

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人羊膜上皮细胞在角膜组织工程中的价值。

人羊膜上皮细胞 (hAEC) 由于易于获得而成为组织工程中潜在的关键参与者之一。本研究的目的是开发一种最佳的分离和运输技术，以及确定 hAECs 的免疫表型和上皮基因表达。从绒毛膜上机械剥离羊膜并用胰蛋白酶-乙二胺四乙酸消化。分离的 hAEC 在含有 10 ng/mL 表皮生长因子的培养基中培养，直至 P4。通过定量聚合酶链反应、流式细胞术和免疫细胞化学分析hAECs的上皮基因表达、细胞表面抗原和蛋白表达。hAECs 也在脂肪生成、成骨和神经源性诱导培养基中培养。在消化 15 片羊膜 (2 × 2 cm) 时观察到 hAEC 的最佳细胞产量，并在用胰蛋白酶消化后 30 分钟分离。F12:Dulbecco 改良的 eagle 培养基是在 4°C 下短期储存的最佳培养基。hAEC 表达 CD9、CD44、CD73 和 CD90，可忽略不计地表达 CD31、CD34、CD45 和 CD117。连续传代后，CK3、CK19 和外皮蛋白基因表达上调，而 p63、CK1 和 CK14 基因表达下调。观察到整合素β1和CK18的持续基因表达。在初始培养时，这些细胞可能具有类似干细胞的特性。然而，它们在连续传代后分化。尽管如此，hAECs 具有上皮干细胞特征，并具有分化为角膜上皮细胞的潜力。体外培养。