Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells
Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells
复制标题
DOI：
10.1167/iovs.16-19805
复制
发表时间：
2016-08-01
影响因子：
4.4
通讯作者：
Hamuro, Junji
中科院分区：
文献类型：
--
作者：
Ueno, Morio;Asada, Kazuko;Hamuro, Junji
目的.我们阐明了一种使用分泌的miRNA谱来鉴定培养的人角膜内皮细胞（cHCECs）适用于细胞注射治疗的方法。cHCEC在其异质亚群（SP）的复合物中的变化与其表面分化簇（CD）标记物有关。利用3D-Gene Human microRNA芯片对培养上清（CS）中的microRNA（miRNA）谱进行综合分析。为了验证3D-Gene结果，从具有不同形态和SP组成的许多培养物进行定量实时PCR。还分析了CS中的外来体和miRNA。在形态多样的cHCEC SP之间分泌的miRNA谱被证明对于个体区分是有用的。用于区分CD 44（-）SP与具有与CD 44（+）表型相似的CD 44（++）SP的候选miRNA是miR 221- 3 p、1246、1915- 3 p和4732- 5 p。与对照培养基相比，在没有细胞状态转换（CST）的cHCEC CS中，后三种miR的水平显著降低，而在具有衰老样CST的cHCEC中，后三种miR的水平显示出增加。MicroR 184的减少与cHCECs上CD 44的上调成反比。在有衰老样CST的cHCEC-CS中，CD 9+外泌体的表达高于无CST的cHCEC-CS，提示这些细胞外囊泡（EVs）可能进入无CST的cHCEC-CS。可通过测量CS中miRNA或外来体的量来区分具有成熟HCEC的CD 44（-）表型的培养的HCEC。因此，CS中的miRNA可以作为鉴定cHCEC的工具。未来通过这些EV对细胞间通信的详细分析可能会为更好地理解HCEC培养物中的CST开辟新的途径。
PURPOSE. We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy.METHODS. The variations of cHCECs in their composites of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers. Integrated analysis of micro RNA (miRNA) profiles in culture supernatants (CS) were investigated by 3D-Gene Human microRNA Chips. To validate 3D-Gene results, quantitative real-time PCR was done from numerous cultures with distinct morphology and SP composition. Exosomes and miRNAs in CS also were analyzed.RESULTS. Secreted miRNA profiles among morphologically-diverse cHCEC SPs proved useful for individual distinction. Candidate miRNAs to discriminate CD44(-) SPs from those with CD44(++) similar to CD44(+++) phenotypes were miRs 221-3p, 1246, 1915-3p, and 4732-5p. The levels of the latter-three miRs decreased dramatically in cHCEC CS without cell-state transition (CST) compared to those of control medium, whereas those from cHCECs with senescence-like CST showed an increase. MicroR184 decreased inversely in parallel with the upregulation of CD44 on cHCECs. CD9+ exosomes were more elevated in cHCEC CS with senescence-like CST than those without CST, indicating the possible import of these extracellular vesicles (EVs) into cHCECs without CST.CONCLUSIONS. Cultured HCECs sharing a CD44(-) phenotype of matured HCECs may be discriminated by measuring the amount of miRNAs or exosome in CS. Thus, miRNA in CS may serve as a tool to qualify cHCECs. Future detailed analysis of cell-to-cell communication via these EVs might open novel pathways for a better understanding of CST in HCEC cultures.