BACKGROUND Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET). METHODS STEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry. RESULTS CD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups. CONCLUSION Human ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

中文翻译：

---

脂肪间充质干细胞衍生的上皮祖细胞对角膜上皮缺损的细胞治疗。

背景技术与角膜缘干细胞缺乏症（LSCD）相关的持续性上皮缺损（PED）需要具有稳定的角膜上皮（CE）的眼表重建。这项研究调查了使用人类脂肪间充质干细胞（ADSC）进行的CE改造，该细胞通过间充质-上皮转化（MET）衍生上皮祖细胞。方法将STEMPRO人ADSC与拮抗糖原合酶激酶3和转化生长因子-β信号转导的特异性抑制剂一起培养，然后在确定的祖细胞靶向上皮分化条件下培养，生成上皮样细胞（MET-Epi）。使用免疫荧光和流式细胞仪对细胞活力，间充质和上皮表型进行表征。纤维蛋白凝胶上的组织工程化（TE）MET-Epi细胞被移植到由碱损伤引起的大鼠LSCD模型的角膜表面。通过荧光素染色，裂隙灯生物显微镜，前节光学相干断层扫描和免疫组织化学评估上皮愈合，角膜浮肿和雾度分级，CE形成。结果CD73high / CD90high / CD105high / CD166high / CD14negative / CD31negative人类ADSC进行MET，获得了表达δNp63，CDH1（E-钙粘蛋白），表皮生长因子受体，整联蛋白-β4和细胞角蛋白（CK）-5的上皮样祖细胞， 9.在明确的上皮分化培养下，这些祖细胞产生表达细胞连接蛋白ZO1和occludin的MET-Epi细胞。当用LSCD诱导的PED移植到大鼠角膜表面时，TE-MET-Epi可以更有效地上皮愈合，与未经治疗或TE-ADSC移植的角膜相比，可抑制角膜水肿和混浊。CE标记（CK3、12和CDH1）在TE-MET-Epi移植的角膜上表达，但在其他对照组中不表达。结论人类ADSC来源的上皮样细胞通过MET从与LSCD相关的PED中回收了CE。ADSC可能是可行的成人干细胞来源，可用于潜在的基于自身上皮细胞的角膜表面疾病治疗。