Lužnik Zala, Hawlina Marko, Maličev Elvira, Bertolin Marina, Kopitar Andreja Nataša, Ihan Alojz, Ferrari Stefano, Schollmayer Petra
Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
Blood Transfusion Centre of Slovenia, Ljubljana. Slovenia.
PLoS One. 2016 Oct 10;11(10):e0164408. doi: 10.1371/journal.pone.0164408. eCollection 2016.
To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).
Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.
Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.
Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.
评估在无任何支架的情况下或在羊膜（AM）上培养的延长时间的角膜缘外植体对假定的表型明确的培养角膜缘间充质干细胞（LMSC）和上皮干细胞（LESC）的活力、增殖及分化潜能的影响。
使用仅添加人血清的培养基，将角膜缘外植体培养在冷冻保存的完整羊膜或塑料板上。羊膜放置时上皮面或基质面朝上。通过流式细胞术对生长出的细胞进行免疫表型分析，检测间充质干细胞标志物（CD73/CD90/CD105阳性且CD45阴性）、增殖和假定祖细胞标志物（CXCR4、CD117）、上皮标志物及抗原呈递细胞标志物（CD80、CD83、CD86）的共表达情况。对在羊膜上培养的角膜缘培养物进行免疫组织化学分析，使用针对泛细胞角蛋白、p63、Ki67的抗体。
形态学和免疫染色分析显示了两种不同的干细胞群体类型，在延长的培养时间段内均可识别。在无羊膜培养的培养物中，LMSC标志物和CXCR4的表达显著更高（p < 0.05）。然而，CD117表达未观察到统计学上的显著差异。在羊膜上培养的细胞保留了上皮细胞结构，组织学检查进一步证实了这一点。组织学显示羊膜两侧有角膜缘上皮生长以及p63、Ki67阳性细胞。
与在塑料培养板上培养的角膜缘细胞相比，在羊膜上培养的角膜缘细胞LMSC和CXCR4标志物的表达谱较低。然而，CD117表达相似。组织学证实羊膜两侧有角膜缘上皮细胞生长，细胞形态无差异，且p63和Ki67呈阳性。
