Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
PLoS One. 2019 Mar 12;14(3):e0212524. doi: 10.1371/journal.pone.0212524. eCollection 2019.
Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function.
Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed.
Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups.
For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.
角膜缘干细胞移植是治疗角膜缘干细胞缺乏症的一种很有前途的疗法。 角膜缘细胞可以从患者眼睛的健康部位或供体的眼睛中采集。 较小的标本不太可能对供体部位造成伤害。 我们研究了角膜缘标本大小对移植功能的多个重要特征的影响。
从大（角膜周长 3 毫米）和小（角膜周长 1 毫米）角膜缘标本中扩增人角膜缘上皮细胞 3 周，并通过光镜、免疫组织化学和透射电镜进行鉴定。 评估上皮厚度、分层、生长、超微结构和表型。
两组间上皮厚度和分层相似。 生长大小与标本大小呈正相关（r = 0.37；P = 0.01），而折叠生长与标本大小呈负相关（r = -0.55；P < 0.0001）。 来自大标本的细胞表达角膜缘上皮细胞标志物 K19 的比例（99.1±1.2%）高于来自小标本的细胞（93.2±13.6%，P = 0.024）。 表达 ABCG2、整合素β1、p63 和 p63α的细胞比例，这些细胞标志物提示不成熟表型；角化蛋白 3、连接蛋白 43 和 E-钙粘蛋白，这些是分化的标志物；以及 Ki67 和 PCNA，这些都表明细胞增殖在两组中是相等的。 两组间桥粒和半桥粒密度相等。
就本研究中使用的供体和培养条件而言，大标本比小标本在生长面积方面更具优势。 就角膜缘上皮细胞厚度、分层、机械强度和获得主要不成熟表型而言，大标本和小标本都是足够的。
