National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Stem Cells Dev. 2011 Sep;20(9):1615-25. doi: 10.1089/scd.2010.0390. Epub 2011 Jan 23.
To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.
为了评估人羊水（HAF）对视网膜色素上皮细胞（RPE）生长和向视网膜神经元转分化的影响，我们从新生儿人尸眼球中分离出 RPE 细胞，并在添加 10%胎牛血清（FBS）的 Dulbecco 改良 Eagle 培养基-F12 中进行培养。将汇合的单层培养物用含 FBS 或 HAF 的培养基进行胰蛋白酶消化和传代。羊水样本取自妊娠早期的孕妇。通过细胞增殖和死亡酶联免疫吸附试验评估 HAF 对 RPE 细胞生长的影响。还使用免疫细胞化学和实时聚合酶链反应技术研究向杆状光感受器和视网膜神经节细胞的转分化。在含 FBS 或 HAF 的培养基中成功建立了 RPE 细胞的原代培养物，导致细胞快速生长和增殖。当 RPE 细胞被转移到体外培养系统时，它们开始失去其分化标志物，如色素沉着和 RPE65 标志物，并通过形态学检测到神经样细胞的球形集落，这些细胞属于干细胞/祖细胞。HAF 处理培养物的免疫细胞化学（ICC）分析显示 Rhodopsin 基因的表达相当高（30% Rhodopsin 阳性细胞），表明 RPE 细胞向杆状光感受器的转分化。实时聚合酶链反应显示 Thy-1 基因（RGC 标志物）的 HAF 剂量依赖性表达，以及 HAF 对 RGC 生成的显著促进作用。这里提供的数据表明，HAF 对 RPE 细胞的生长和向视网膜神经元的转分化具有宝贵的刺激作用。它可以被视为无血清种类的神经视网膜再生研究中使用的培养基中引入的新的富含补充物。
