Eye Center, Medical Center-Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany.
Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany.
Int J Mol Sci. 2022 Mar 2;23(5):2750. doi: 10.3390/ijms23052750.
The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117CD90), LM (CD117CD90), and LEPC (CD117CD90). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4-5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63 and Ki67 cells and decreased CK12 cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.
人眼角膜缘的上皮祖细胞（LEPC）的命运决定于周围的微环境，其中角膜缘巢细胞（LNC）是其重要组成部分之一。对新鲜分离的 LNC 的研究主要包括角膜缘间充质基质细胞（LMSC）和角膜缘黑素细胞（LM），但由于缺乏有效分离和纯化这些细胞的方案，研究受到了阻碍。我们设计了一种通过胶原酶消化角膜缘组织并随后使用针对 CD90 和 CD117 的荧光激活细胞分选（FACS）快速获取纯 LMSC、LM 和 LEPC 群体的方案。通过免疫表型和功能测定对分选细胞进行了表征。在 3D 共培养中研究了 LMSC 和 LM 对 LEPC 的影响，并通过免疫组织化学评估了 LEPC 的分化状态。酶消化和流式细胞分选产生了纯 LMSC（CD117CD90）、LM（CD117CD90）和 LEPC（CD117CD90）群体。LMSC 具有自我更新能力（55.0±4.6 群体倍增），表达间充质干细胞标志物（CD73、CD90、CD105 和 CD44），并向脂肪细胞、成骨细胞或软骨细胞分化。LM 具有自我更新能力和持续的黑色素产生能力。分选的 LEPC 表达上皮祖细胞标志物（CK14、CK19 和 CK15），并具有集落形成能力。LMSC 和 LM 与 LEPC 的共培养导致 4-5 层分层上皮，并支持 LEPC 表型的保存，与 LEPC 单核培养相比，p63 和 Ki67 细胞增加，CK12 细胞减少。从单个制备物中高效分离纯 LM、LMSC 和 LEPC 群体可直接进行转录组和蛋白质组谱分析以及对天然未传代 LNC 的功能研究，可将其视为体内 LNC 的适当等效物。所开发的仿生 3D 共培养方法可为研究 LNC 在角膜缘干细胞巢中的功能作用提供实验模型。
