Novel Identity and Functional Markers for Human Corneal Endothelial Cells.
Novel Identity and Functional Markers for Human Corneal Endothelial Cells.
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DOI：
10.1167/iovs.15-18826
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发表时间：
2016-05-01
影响因子：
4.4
通讯作者：
Goldberg JL
中科院分区：
文献类型：
--
作者：
Bartakova A;Alvarez-Delfin K;Weisman AD;Salero E;Raffa GA;Merkhofer RM Jr;Kunzevitzky NJ;Goldberg JL
人角膜内皮细胞 (HCEC) 密度随着年龄、手术并发症或疾病而降低，导致视力障碍。尽管全球范围内缺乏移植级组织，但这种内皮功能障碍是角膜移植的指征。为了克服目前供体供应不足的问题，我们在体外分离、扩增和表征 HCEC，作为细胞治疗的一步。从尸体角膜中分离出人角膜内皮细胞并在体外扩增。基于形态学和免疫细胞化学评估细胞身份，并使用基因表达分析和流式细胞术来鉴定新的 HCEC 特异性标记物。通过跨内皮电阻 (TEER) 测定评估 HCEC 形成屏障的功能能力。培养的 HCEC 在最多四代中表现出典型的形态，随后经历了内皮-间质转化 (EnMT)。供体组织的质量影响培养中的细胞测量，包括增殖率。培养的 HCEC 表达身份标记，微阵列分析揭示了新的内皮特异性标记，并通过流式细胞术进行了验证。最后，典型 HCEC 表达更高水平的 CD56，这与比成纤维细胞 HCEC 更高的 TEER 相关。来自尸体供体角膜的 HCEC 的体外扩增产生了可通过形态和一组新标记物识别的功能细胞。所描述的标记物与培养物中的功能相关，这表明了角膜内皮功能障碍的细胞疗法的基础。
Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.