Cultured Human Corneal Endothelial Cell Aneuploidy Dependence on the Presence of Heterogeneous Subpopulations With Distinct Differentiation Phenotypes
Cultured Human Corneal Endothelial Cell Aneuploidy Dependence on the Presence of Heterogeneous Subpopulations With Distinct Differentiation Phenotypes
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DOI：
10.1167/iovs.16-19771
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发表时间：
2016-08-01
影响因子：
4.4
通讯作者：
Kinoshita, Shigeru
中科院分区：
文献类型：
--
作者：
Hamuro, Junji;Ueno, Morio;Kinoshita, Shigeru
目的。培养的人角膜内皮细胞有望成为治疗角膜内皮功能障碍的供体角膜的替代物。然而，据报道，cHCECs在体外细胞分裂过程中往往会出现染色体异常，从而阻碍了它们在临床上的应用。本研究的目的是阐明特定亚群的异种cHCECs是否会出现异倍体，而其他亚群则不会。方法采用流式细胞术检测cHCECs表面分化簇(CD)抗原CD166、CD105、CD44、CD26和CD24的表达水平，分析异种cHCECs中SPS的存在。对23批cHCECs进行了细胞遗传学检查，其中包括由混合SP组成的全细胞制剂(批量)和磁激活细胞分选(MACS)半纯化的SP。HCEC供者年龄从9岁到69岁，培养代次从原代到第五代。结果：流式细胞术分析显示至少存在三种cHCEC SP。经MACS纯化的1个SP表面表达CD166(+)、CD105(-)、CD44(-)、CD24(-)、CD26(-)，50个细胞中未见异倍体。CD166(+)、CD44(+)、CD24(-)和CD26(+)cHCEC SPS在所有细胞(60个细胞)中均表现为性染色体丢失，而CD166(+)、CD44(+)、CD24(+)和CD26(-)SPS在第6、7、12和20号染色体上虽有部分三体，但CD166(+)、CD44(+)、CD24(+)和CD26(-)SPS在所有细胞(60个细胞)中均表现为性染色体丢失。
PURPOSE. Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, cHCECs reportedly tend to exhibit chromosomal abnormality during in vitro cell division, thereby hampering their use in the clinical setting. The purpose of this study was to clarify whether a specified subpopulation (SP) of heterogeneous cHCECs would exhibit aneuploidy, whereas other SPs would not.METHODS. The presence of SPs in cHCECs was analyzed on the basis of surface cluster of differentiation (CD) antigen CD166, CD105, CD44, CD26, and CD24 expression levels by flow cytometry. Cytogenetic examination was performed for 23 lots of cHCECs, either as whole-cell preparations (bulk) consisting of mixed SPs or as a semipurified SP by magnetic activated cell sorting (MACS). The HCEC donors ranged from 9 to 69 years of age and the culture passages from primary to fifth passage.RESULTS. Flow cytometry analysis demonstrated the presence of at least three cHCEC SPs. One SP, purified by MACS, with surface expression of CD166(+), CD105(-), CD44(-), CD24(-), and CD26(-) did not show any aneuploidy in 50 cells. However, CD166(+), CD44(+++), CD24(-), and CD26(+) cHCEC SPs showed sex chromosome loss in all cells (60 cells), whereas CD166(+), CD44(+++), CD24(+), and CD26(-) SPs exhibited, albeit partly, trisomy on chromosomes 6, 7, 12, and 20.CONCLUSIONS. We found that cHCEC aneuploidy is linked to specified SPs present in cHCECs and that the SP sharing the surface phenotype with mature HCECs in corneal tissues was devoid of the karyotype abnormality.