Meller D, Pires R T F, Tseng S C G
Department of Ophthalmology, Bascom Palmer Eye Institute, USA.
Br J Ophthalmol. 2002 Apr;86(4):463-71. doi: 10.1136/bjo.86.4.463.
BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo.
The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins.
Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C.
These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.
背景/目的：羊膜（AM）移植可有效扩增部分角膜缘干细胞缺乏患者剩余的角膜缘上皮干细胞。作者研究了这种作用是否能在体外产生。
比较了源自人角膜缘、周边角膜和中央角膜的外植体在羊膜上的生长率。对于人角膜缘上皮细胞（HLEC）的生长，通过BrdU标记1天或7天来测量细胞周期动力学，其中7天标记的样本在原代培养、二代3T3成纤维细胞培养以及用佛波酯短暂处理后的无胸腺Balb/c小鼠中进行追踪。通过组织学和透射电子显微镜研究上皮形态，并用针对角蛋白和粘蛋白的单克隆抗体进行免疫染色来定义表型。
中央角膜和周边角膜外植体的生长率分别为0/22（0%）和2/24（8.3%），而角膜缘外植体的生长率为77/80（96.2%）（p<0.0001）。24小时BrdU标记显示，65%的角膜缘外植体标记指数始终较低（即低于5%），但35%的角膜缘外植体呈现混合模式，部分区域标记指数较高（即高于40%），而所有（100%）周边角膜外植体均如此。连续7天进行BrdU标记发现，61.5%的角膜缘外植体标记指数较高，其余仍保持较低标记指数。在原代培养中进行7天标记后再追踪14天，或移植到3T3成纤维细胞饲养层后追踪21天，发现有许多标记保留细胞。在用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯处理24小时并进行7天标记后，移植到无胸腺小鼠体内的HLEC在追踪9天后仍显示出一些标记保留的基底细胞。在羊膜上培养的HLEC对K14角蛋白和MUC4呈强阳性，基底上层细胞对K3角蛋白呈弱阳性，而对K12角蛋白、AMEM2和MUC5AC呈阴性。在无胸腺小鼠皮下植入后，形成的上皮明显分层，基底上皮细胞对K14角蛋白呈强阳性，基底上层上皮细胞对K3角蛋白和MUC4呈强阳性，整个上皮对K12角蛋白和MUC5A/C呈阴性。
这些数据支持以下观点，即羊膜培养可优先保存和扩增角膜缘上皮干细胞，这些干细胞保留了其在体内的慢周期、标记保留和未分化特性。这一发现支持了利用少量供体角膜缘组织在体外扩增角膜缘上皮干细胞以治疗完全角膜缘干细胞缺乏患者的可行性。
