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Identification and purification of retinal stem cells by the expression pattern of specific genes.

通过特定基因的表达模式鉴定和纯化视网膜干细胞。

基本信息

批准号：
15390088
负责人：
WATANABE Sumiko
金额：
$ 9.86万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Neural retina is an important target organ for regenerative medicine, and isolation and expansion of retinal progenitor cells are critical issues from both scientific and clinical views. However, the characters of the immature retinal cells are not elucidated because of the lack of prospective approach to identify retinal progenitor cells.We screened the expression pattern of cell surface proteins in mouse immature retina by flow cytometer using about 120 antibodies against different membrane proteins. Among them, 20 antibodies recognized sub-populations of immature retina, and we examined the proliferation and differentiation abilities of purified those sub-populations of retina.We identify the SSEA-1 (CD15), which is expressed in ES cells and neural stem cells, as a novel surface antigen to define immature retinal progenitor cells. SSEA-1 positive cells are in the peripheral region of retina of the E17 embryo and then dramatically disappeared along with retinal development. SSEA-1 st … More rong positive cells were Ki-67 antigen positive and had prolonged proliferation activities than that of SSEA-1 negative cells in reaggregation culture. Moreover, differentiation of SSEA-1 cells into late born retinal subtypes took longer period, suggesting that these cells are at more immature stage than SSEA-1 negative cells.To obtain new cell markers defining retinal stem cells, we took an approach of proteomics. Membrane fractions were purified from embryonic and adult retina, and the profile of the expression pattern of membranous proteins were examined by shotgun analyses on a nanoflow LC-MS/MS system. Several proteins which were expressed specifically in embryo or adult retina were identified, and biological functions of these proteins are currently examined by si-RNA.Taken together, it is the first report showing the finding of a surface marker to determine regionally restricted immature subset of progenitor cells of neural retina and may serve an excellent tool for clinical use of retinal progenitor cells. Less

神经视网膜是再生医学的重要靶器官，视网膜祖细胞的分离和扩增是科学和临床上的关键问题。然而，由于缺乏前瞻性的方法来鉴定视网膜前体细胞，因此未成熟视网膜细胞的特征尚未阐明，我们使用约120种针对不同膜蛋白的抗体，通过流式细胞仪筛选小鼠未成熟视网膜细胞表面蛋白的表达模式。其中20种抗体可识别未成熟视网膜的亚群，我们检测了纯化的这些亚群视网膜的增殖和分化能力。我们鉴定了在ES细胞和神经干细胞中表达的SSEA-1（CD 15）作为一种新的表面抗原来定义未成熟视网膜祖细胞。SSEA-1阳性细胞分布于E17胚胎视网膜的周边区域，随着视网膜的发育沿着消失。SSEA-1 ...更多信息在再聚集培养中，SSEA-1阳性细胞Ki-67抗原阳性，增殖活性明显高于SSEA-1阴性细胞。此外，SSEA-1细胞分化为晚生视网膜干细胞所需的时间较长，提示这些细胞比SSEA-1阴性细胞处于更不成熟的阶段。从胚胎和成人视网膜中纯化膜组分，并在纳米流LC-MS/MS系统上通过鸟枪法分析来检查膜蛋白的表达模式。本研究首次发现了一种可用于神经视网膜前体细胞定位的表面标记物，为视网膜前体细胞的临床应用提供了一个良好的工具。少