Szabó Dóra Júlia, Nagymihály Richárd, Veréb Zoltán, Josifovska Natasha, Noer Agate, Liskova Petra, Facskó Andrea, Moe Morten C, Petrovski Goran
Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Center of Eye Research, Department of Ophthalmology, Oslo University Hospital, University of Oslo, Oslo, Norway.
Histol Histopathol. 2018 May;33(5):455-462. doi: 10.14670/HH-11-928. Epub 2017 Sep 5.
Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder.
Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours.
CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively.
MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.
多层小体（MLBs）是通过自噬依赖性机制形成的同心细胞质膜。在角膜中，MLBs的存在与施奈德角膜营养不良（SCD）相关。角膜基质和SCD的体外三维建模有助于研究该疾病的发病机制和解决方法。
从尸体中分离角膜基质外植体，并长期培养超过3个月，以实现角膜基质来源的间充质干细胞样细胞（CSMSCs）的自发三维生长。然后通过透射电子显微镜（TEM）检查三维组织中MLBs的存在情况，并通过针对自噬标志物（p62、LC3）的免疫荧光标记进行检测。通过经典的血清饥饿或雷帕霉素（RAP）处理（50 nM）诱导自噬，并通过自噬抑制剂3-甲基腺嘌呤（3-MA，10 mM）抑制24小时。
CSMSCs可在3-4周内自发形成三维生长物，沉积含有I型胶原蛋白的自身细胞外基质。TEM证实长期（>3个月）三维培养物中存在MLBs，在饥饿和RAP处理下MLBs变得更加丰富，而在3-MA抑制自噬时数量减少。自噬的存在及其消失可分别通过LC3和p62表达的反向相关增加和减少来证实。
长期CSMSC培养物中MLB的形成可作为研究包括SCD在内的角膜基质疾病的潜在体外模型。抑制自噬可减少MLBs的形成，这可能在未来导致该疾病的新治疗方法。
