Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma
Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma
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DOI：
10.1167/iovs.07-0587
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发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Funderburgh, James L.
中科院分区：
文献类型：
--
作者：
Du, Yiqin;SundarRaj, Nirmala;Funderburgh, James L.
目的.目的探讨人角膜基质干细胞在体外向角膜基质细胞表型转化并组织细胞外基质（ECM）的潜能。采用流式细胞术分离人角膜基质干细胞（hCSSC）作为侧群细胞。将克隆的hCSSC在无血清培养基中作为自由漂浮的沉淀培养3周。采用基因阵列、定量RT-PCR、免疫染色和免疫印迹检测基因表达。透射电子显微镜显示胶原纤维大小和扩张。无血清培养基中的hCSSC颗粒培养物上调了角质细胞特异性基因的表达，并分泌了大量含有特征性基质成分的ECM：角质蛋白聚糖、硫酸角质素、胶原蛋白I、胶原蛋白V和胶原蛋白VI。颗粒中丰富的连接蛋白43和钙粘蛋白11表明了体内角膜细胞典型的细胞-细胞连接。颗粒培养物的电子显微镜显示丰富的纤维状胶原蛋白，其中一些是在平行阵列排列类似于那些基质lamicum。基因阵列鉴定了角膜基质细胞高度表达的几种基因的颗粒表达。与hCSSC相比，这些角膜细胞基因FLJ 30046、KERA、ALDH 3A 1、CXADR、PTGDS、PDK 4、MTAC 2D 1、F13 A1-的转录物在颗粒中增加多达100倍。同时，与hCSSC相比，干细胞基因BMI 1、KIT、NOTCH 1、SIX 2、PAX 6、ABCG 2、SPAG 10和OSIL的表达在颗粒中以相似的因子降低。hCSSC的无支架沉淀培养诱导这些细胞中的角膜细胞基因表达模式和有组织的基质样ECM的分泌。这些细胞为角膜生物工程提供了新的潜力。
PURPOSE. To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix ( ECM) in vitro similar to corneal stromal tissue.METHODS. Human corneal stromal stem cells ( hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment.RESULTS. Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1 - were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC.CONCLUSIONS. Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.