Regenerative Medicine Group, Reliance Life Sciences Pvt. Ltd., R-282, TTC Area of MIDC, Thane-Belapur Road, Rabale, Navi Mumbai 400 701, India.
Exp Eye Res. 2011 Jan;92(1):47-56. doi: 10.1016/j.exer.2010.11.001. Epub 2010 Nov 18.
Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin β1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.
异体来源的体外扩增自体角膜缘干细胞移植已在全球范围内应用于治疗角膜缘干细胞缺乏症（LSCD）患者。然而，对于单侧 LSCD 患者，从正常眼分离角膜缘组织仍然是供体的主要关注点。更重要的是，对于双侧 LSCD 患者，自体细胞移植不是一种可行的选择。本研究的目的是确定人角膜缘上皮干细胞（hLESCs）的扩增潜力，以便将来可能用于异体移植。共培养和扩增了 6 例角膜缘活检样本，直到传代 1 代（P-1），此时将 hLESCs 冷冻保存。半定量 RT-PCR 和免疫表型分析表明，冷冻保存前后获得的 hLESCs 保留了主要角膜缘上皮干细胞标志物的表达，如 p63、SSEA-4、ABCG2、细胞角蛋白 19（CK19）、整合素β1 和波形蛋白。一个显著的区别是，P-0 hLESCs 表达 HLA-DR mRNA，而扩增和冷冻保存的样本则没有 HLA-DR 基因表达。人 LESCs 不表达共刺激蛋白 CD80 或 B7-DC，但在细胞表面表达显著水平的 CD86、B7-H1 和 HLA-ABC 分子。IFN-γ 处理 hLESCs 诱导这些细胞表达 HLA-DR、吲哚胺 2,3-双加氧酶（IDO）和 HLA-G。即使存在促炎细胞因子 IFN-γ，体外培养的 hLESCs 也不能刺激同种异体 T 细胞增殖。这些结果表明，冷冻保存的 hLESCs 本质上无免疫原性，并表达负免疫调节分子，这对于它们在同种异体环境中的存活可能至关重要。
