Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue
Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue
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DOI：
10.1167/iovs.14-16246
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发表时间：
2015-11-01
影响因子：
4.4
通讯作者：
Stern, Jeffrey H.
中科院分区：
文献类型：
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作者：
Blenkinsop, Timothy A.;Saini, Janmeet S.;Stern, Jeffrey H.
目的.我们用一种新的成人RPE培养方案测试了保留了哪些天然特征。我们从成年人的眼睛培养RPE。采用免疫组织化学、电子显微镜、电生理学、液体转运和ELISA的标准方案。成人RPE培养物的融合单层表现出天然RPE的特征。免疫组织化学显示RPE标记物的两极表达。电子显微镜显示了天然RPE的特征。平均跨上皮电位（TEP）为1.19 +/- 0.24 mV（平均值+/- SEM，n = 31），心尖阳性，平均跨上皮电阻（R-T）为178.7 +/- 9.9 Omega.cm（2）（平均值+/- SEM，n = 31）。施用100 μ M三磷酸腺苷（ATP）可使净液体吸收（Jv）增加6.11 +/- 0.53 μ L.cm（2）。h（-1）（平均值+/- SEM，n = 6）和TEP 0.33 +/- 0.048 mV（平均值+/- SEM，n = 25）。培养的RPE的基因表达与天然成人RPE（n = 5）相当;然而，天然RPE RNA在死亡后24至40小时之间收获，因此可能不能准确反映健康的天然RPE。血管内皮生长因子在基底部优先分泌2582 ± 146 pg/mL/d，而在心尖部优先分泌1548 ± 162 pg/mL/d（n = 14，P < 0.01），而PEDF优先在顶侧分泌1487 +/- 280 ng/mL/d，而基底侧分泌864 +/- 132 ng/mL/d（n = 14，P < 0.01）。新的培养模型保留了天然RPE的形态学、电生理学以及基因和蛋白质表达模式，并且可能是研究RPE生理学、疾病和移植的有用模型。
PURPOSE. We tested what native features have been preserved with a new culture protocol for adult human RPE.METHODS. We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.RESULTS. Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 +/- 0.24 mV (mean +/- SEM, n = 31), apical positive, and the mean transepithelial resistance (R-T) was 178.7 +/- 9.9 Omega.cm(2) (mean +/- SEM, n = 31). Application of 100 mu M adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 +/- 0.53 mu L.cm(2). h(-1) (mean +/- SEM, n = 6) and TEP by 0.33 +/- 0.048 mV (mean +/- SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 6 146 pg/mL/d, compared to an apical secretion of 1548 +/- 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 +/- 280 ng/mL/d compared to a basolateral secretion of 864 +/- 132 ng/mL/d (n = 14, P < 0.01).CONCLUSIONS. The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.