Braunsperger Moritz Vincent, Martin Gottfried, Herzig Tabea, Kußberger Isabell, Gießl Andreas, Steimle Stefan, Schlötzer-Schrehardt Ursula, Schlunck Günther, Reinhard Thomas, Polisetti Naresh
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 Erlan-gen-Nürnberg, Schwabachanlage 6, D-91054, Erlangen, Germany.
Stem Cell Rev Rep. 2025 Apr 16. doi: 10.1007/s12015-025-10877-w.
Limbal epithelial stem/progenitor cells (LEPC), supported by limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) within a specialized niche, are responsible for maintaining the corneal epithelium. Small extracellular vesicles (sEV) emerged as critical mediators of intercellular communication in various stem cell niches, yet their role in maintaining human limbal niche homeostasis remains poorly understood. In this study, tangential flow filtration and size exclusion chromatography were used to isolate sEV from LEPC-, LMSC- and LM-conditioned media. The isolated sEV from LEPC exhibited properties characteristic for sEV as confirmed by nanoparticle tracking analysis for size and concentration, by electron microscopy for morphology, and by western blot analysis of canonical EV markers including the cell-specific protein (cytokeratin 17/19). Quantitative and comparative proteomic profiling revealed distinct molecular signatures of LEPC-derived sEV, enriched in factors associated with keratinocyte development, extracellular matrix organization, and niche regulation. These findings suggest that LEPC-derived sEV may serve as important signaling mediators within the limbal niche microenvironment, though additional studies are needed to determine their specific functional roles in maintaining niche homeostasis.
角膜缘上皮干细胞/祖细胞（LEPC）在一个特殊的微环境中，受到角膜缘间充质基质细胞（LMSC）和角膜缘黑素细胞（LM）的支持，负责维持角膜上皮。小细胞外囊泡（sEV）已成为各种干细胞微环境中细胞间通讯的关键介质，但其在维持人角膜缘微环境稳态中的作用仍知之甚少。在本研究中，采用切向流过滤和尺寸排阻色谱法从LEPC、LMSC和LM条件培养基中分离sEV。通过纳米颗粒跟踪分析测定大小和浓度、通过电子显微镜观察形态以及通过对包括细胞特异性蛋白（细胞角蛋白17/19）在内的典型EV标志物进行蛋白质印迹分析，证实从LEPC分离的sEV具有sEV的特征性质。定量和比较蛋白质组学分析揭示了LEPC衍生的sEV的独特分子特征，富含与角质形成细胞发育、细胞外基质组织和微环境调节相关的因子。这些发现表明，LEPC衍生的sEV可能作为角膜缘微环境中的重要信号介质，不过还需要进一步研究来确定它们在维持微环境稳态中的具体功能作用。
