Ocular Surface Group, IOBA-Institute for Applied Ophthalmobiology, University of Valladolid, Valladolid, Spain.
Curr Eye Res. 2013 Sep;38(9):933-44. doi: 10.3109/02713683.2013.802809. Epub 2013 Jun 14.
Transplantation of autologous corneal stem cells in not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore the ocular surface in these patients, an autologous extraocular source of stem cells is desirable to avoid dependence on deceased donor tissue and host immunosuppression of allogenic transplants. While bone marrow-derived mesenchymal stem cells (MSCs) can acquire certain characteristics of corneal epithelial cells, subcutaneous adipose tissue (AT) is more readily available and accessible. The aim of this study was to determine if extraocular human AT-derived MSCs (hAT-MSCs) can acquire in vitro some features of corneal epithelial-like cells.
hAT-MSCs were isolated from human lipoaspirates and expanded up to 3-4 passages. We studied the immunophenotype of MSCs and demonstrated its multipotent capacity to differentiate toward osteoblasts, adipocytes and chondrocytes. To test the capacity of differentiation of hAT-MSCs toward corneal epithelial-like cells, hAT-MSCs were cultured on substrata of plastic or collagen IV. We used basal culture medium (BM), BM conditioned with human corneal epithelial cells (HCEcBM) and BM conditioned with limbal fibroblasts (LFcBM).
The hAT-MSCs incubated for 15 days with HCEcBM acquired more polygonal and complex morphology as evaluated by phase-contrast microscopy and flow cytometry. Additionally, the expression of transforming growth factor-β receptor CD105 and corneal epithelial marker CK12 got increased as evaluated by flow cytometry, real-time reverse-transcription polymerase chain reaction, western blot and immunostaining. These changes were absent in hAT-MSCs incubated with unconditioned BM or with LFcBM.
Corneal epithelial-like cells can be induced from extraocular hAT-MSCs by subjecting them to an in vitro microenvironment containing conditioning signals derived from differentiated human corneal epithelial cells. Our results suggest that hAT-MSCs could provide a novel source of stem cells that hold the potential to restore sight lost in patients suffering from bilateral ocular surface failure due to LSCD.
在双侧角膜缘干细胞缺乏症（LSCD）的情况下，自体角膜干细胞移植是不可能的。为了恢复这些患者的眼表面，需要一种自体眼外干细胞来源，以避免依赖于已故供体组织和同种异体移植的宿主免疫抑制。虽然骨髓间充质干细胞（MSCs）可以获得角膜上皮细胞的某些特征，但皮下脂肪组织（AT）更容易获得和利用。本研究旨在确定眼外源性人 AT 衍生间充质干细胞（hAT-MSCs）是否可以在体外获得某些角膜上皮样细胞的特征。
从人脂肪抽吸物中分离 hAT-MSCs 并扩增至 3-4 代。我们研究了 MSC 的免疫表型，并证明了其向成骨细胞、脂肪细胞和成软骨细胞分化的多能性。为了测试 hAT-MSCs 向角膜上皮样细胞分化的能力，将 hAT-MSCs 培养在塑料或胶原 IV 基底上。我们使用基础培养基（BM）、用人角膜上皮细胞（HCEcBM）条件化的 BM 和用角膜缘成纤维细胞（LFcBM）条件化的 BM。
用 HCEcBM 孵育 15 天的 hAT-MSCs 通过相差显微镜和流式细胞术评估获得更多边形和复杂的形态。此外，通过流式细胞术、实时逆转录聚合酶链反应、Western blot 和免疫染色评估，转化生长因子-β受体 CD105 和角膜上皮标志物 CK12 的表达增加。在未用未条件化的 BM 或 LFcBM 孵育的 hAT-MSCs 中未观察到这些变化。
通过将 hAT-MSCs 置于含有来自分化的人角膜上皮细胞的调节信号的体外微环境中，可以诱导产生角膜上皮样细胞。我们的结果表明，hAT-MSCs 可以提供一种新的干细胞来源，有可能恢复因 LSCD 导致双眼表面衰竭而丧失视力的患者的视力。
