Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin.
Invest Ophthalmol Vis Sci. 2013 Oct 17;54(10):6767-78. doi: 10.1167/iovs.13-11943.
To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures.
Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE).
RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels.
Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
确定连续扩增对人诱导多能干细胞（hiPSC）衍生的 RPE 培养物的细胞、分子和功能特性的影响。
从四个人体获得的成纤维细胞被重编程为 hiPSC，并使用先前描述的方法分化为 RPE 细胞。深色素沉着的 hiPSC-RPE 斑块被解剖、分离并在培养中生长，直到它们重新形成色素沉着的单层。通过重复的 RPE 分离、扩增和成熟，获得后续的传代。比较不同传代的 hiPSC-RPE 的基因和蛋白表达谱以及形态和功能特征与人类胎儿 RPE（hfRPE）。
当作为色素沉着的单层培养物进行连续传代时，来自四个 hiPSC 系的 RPE 可以扩增超过 1000 倍。重要的是，在前三代（P1-P3）中，hiPSC-RPE 单层的扩增导致多能性和神经视网膜标志物的表达减少，同时保持特征性的形态特征和基因及蛋白表达谱。此外，P1 到 P3 的 hiPSC-RPE 单层能够可靠地显示功能性紧密连接、G 蛋白偶联受体介导的钙瞬变、光感受器外节的吞噬和降解以及生物分子的极化分泌。相比之下，P4 的 hiPSC-RPE 细胞无法形成单层，并且具有改变的形态和功能特征以及基因表达水平。
高度分化的、色素沉着的 hiPSC-RPE 单层可以进行有限的连续扩增，同时保留关键的细胞学和功能特征。然而，传代 hiPSC-RPE 培养物超过衰老会导致失去这些特征。我们的发现支持有限的、受控的传代患者特异性 hiPSC-RPE，以获得用于体外疾病建模、药物筛选和细胞移植所需的细胞。
