Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium
Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium
复制标题
DOI：
--
复制
发表时间：
2012-03
期刊：
影响因子：
2.2
通讯作者：
N. Joyce;D. Harris;Vladimir Markov;Zhe Zhang;Biagio Saitta
中科院分区：
文献类型：
--
作者：
N. Joyce;D. Harris;Vladimir Markov;Zhe Zhang;Biagio Saitta
目的探讨脐带血间充质干细胞（UCB MSCs）向人角膜内皮细胞（HCEC）表型转化的可行性，并通过离体角膜创伤模型观察UCB MSCs能否“归巢”于角膜内皮细胞损伤部位。方法从脐血MSCs和HCECs中分离纯化RNA。通过微阵列分析获得关于UCB MSC和HCEC的相对基因表达的基线信息。定量实时PCR（q-PCR）验证了基因子集的微阵列结果。在组织培养和离体角膜内皮伤口模型中使用三种不同的培养基测试不同培养基引导UCB MSC朝向更HCEC样表型的能力：MSC基础培养基（MSCBM）、用于培养透镜上皮细胞的基础培养基（LECBM）或透镜上皮细胞条件培养基（LECCM）。通过相差显微镜或结晶紫染色的细胞的光镜观察观察MSC的形态。免疫定位的连接相关的蛋白质，zonula occludins-1（ZO 1）和N-钙粘蛋白，可视化荧光共聚焦显微镜。通过用钙螯合剂EGTA处理来测试细胞-细胞连接的形成。第二个微阵列分析比较了在LECBM和LECCM中生长的UCB MSC之间的基因表达，以鉴定由透镜上皮细胞条件培养基诱导的变化。在离体角膜内皮伤口中使用ZO 1免疫定位模式确定UCB MSC“归巢”内皮损伤区域的能力。结果基线微阵列分析提供了UCB MSCs和HCECs中相关基因表达的信息。在离体角膜伤口中，MSC附着于受损但不完整的角膜内皮。当细胞在LECCM存在下生长时，MSC的形态发生一致性改变。在组织培养和离体角膜伤口中，用LECCM处理的UCB MSC被拉长并形成紧密贴壁的平行细胞片。在组织培养和离体角膜内皮伤口中，ZO 1和N-cadherin主要定位于存在MSCBM的UCB MSC的细胞质中。然而，这两种蛋白质定位于细胞边界时，UCB间充质干细胞生长在LECBM或LECCM。这种定位丢失时，细胞外钙水平降低治疗与EGTA。第二个微阵列分析表明，当UCB间充质干细胞在LECCM而不是LECBM中生长时，一个基因子集的相对表达显着不同，提示更HCEC样表型。结论脐血间充质干细胞能够“归巢”于角膜内皮损伤区，并可向HCEC样细胞表型转化。需要进一步的研究来确定特定的微环境条件，这将允许组织工程的脐带血间充质干细胞，以取代受损或患病的角膜内皮。
Purpose To test the feasibility of altering the phenotype of umbilical cord blood mesenchymal stem cells (UCB MSCs) toward that of human corneal endothelial cells (HCEC) and to determine whether UCB MSCs can “home” to sites of corneal endothelial cell injury using an ex vivo corneal wound model. Methods RNA was isolated and purified from UCB MSCs and HCECs. Baseline information regarding the relative gene expression of UCB MSCs and HCEC was obtained by microarray analysis. Quantitative real-time PCR (q-PCR) verified the microarray findings for a subset of genes. The ability of different culture media to direct UCB MSCs toward a more HCEC-like phenotype was tested in both tissue culture and ex vivo corneal endothelial wound models using three different media: MSC basal medium (MSCBM), a basal medium used to culture lens epithelial cells (LECBM), or lens epithelial cell-conditioned medium (LECCM). Morphology of the MSCs was observed by phase-contrast microscopy or by light microscopic observation of crystal violet-stained cells. Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy. Formation of cell-cell junctions was tested by treatment with the calcium chelator, EGTA. A second microarray analysis compared gene expression between UCB MSCs grown in LECBM and LECCM to identify changes induced by the lens epithelial cell-conditioned culture medium. The ability of UCB MSCs to “home” to areas of endothelial injury was determined using ZO1 immunolocalization patterns in ex vivo corneal endothelial wounds. Results Baseline microarray analysis provided information regarding relative gene expression in UCB MSCs and HCECs. MSCs attached to damaged, but not intact, corneal endothelium in ex vivo corneal wounds. The morphology of MSCs was consistently altered when cells were grown in the presence of LECCM. In tissue culture and in ex vivo corneal wounds, UCB MSC treated with LECCM were elongated and formed parallel sheets of closely apposed cells. In both tissue culture and ex vivo corneal endothelial wounds, ZO1 and N-cadherin localized mainly to the cytoplasm of UCB MSCs in the presence of MSCBM. However, both proteins localized to cell borders when UCB MSCs were grown in either LECBM or LECCM. This localization was lost when extracellular calcium levels were reduced by treatment with EGTA. A second microarray analysis showed that, when UCB MSCs were grown in LECCM instead of LECBM, the relative expression of a subset of genes markedly differed, suggestive of a more HCEC-like phenotype. Conclusions Results indicate that UCB MSCs are able to “home” to areas of injured corneal endothelium and that the phenotype of UCB MSCs can be altered toward that of HCEC-like cells. Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.