High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells
High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells
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DOI：
10.1634/stemcells.2005-0311
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发表时间：
2006-09-01
期刊：
影响因子：
5.2
通讯作者：
Arsenijevic, Yvan
中科院分区：
文献类型：
--
作者：
Merhi-Soussi, Faten;Angenieux, Brigitte;Arsenijevic, Yvan
本工作的目的是从视网膜干细胞（RSCs）中产生大量向感光细胞命运定向的细胞，以便为神经发生和移植研究提供无限的细胞来源。我们扩增了具有放射状胶质细胞特征的RSC（至少34代），并在体外用成纤维细胞生长因子（FGF）-2预处理细胞5天，之后用B27补充剂处理细胞以诱导细胞分化和成熟。分化后，细胞表达对应于神经元和神经胶质的细胞类型特异性标记物。我们通过免疫细胞化学分析表明，分化细胞的亚群致力于光感受器谱系，这些细胞以相同的比例表达光感受器蛋白recoverin，peripherin和视紫红质。此外，在分化过程中用表达绿色荧光蛋白（GFP）的慢病毒载体感染的细胞在视紫红质启动子或光感受器间类视色素结合蛋白（IRBP）启动子的控制下表达GFP。FGF-2引发增加神经元分化，同时减少胶质细胞的产生。逆转录-聚合酶链反应分析显示，分化的细胞表达光受体特异性基因，如Crx，视紫红质，外周蛋白，IRBP和磷酸二酯酶-α。分化细胞的定量显示出向光感受器谱系的稳健分化：约25%-35%的总细胞具有光感受器标志物。大量未分化的RSC以及分化的光感受器的产生将使研究人员能够通过移植研究确定哪些细胞最适合整合退化的视网膜。
The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells and primed the cells in vitro with fibroblast growth factor (FGF)-2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type-specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage given that these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing green fluorescent protein (GFP) under the control of either the rhodopsin promoter or the interphotoreceptor retinoid-binding protein (IRBP) promoter, expressed GFP. FGF-2 priming increased neuronal differentiation while decreasing glia generation. Reverse transcription-polymerase chain reaction analyses revealed that the differentiated cells expressed photoreceptor-specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase-alpha. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: Approximately 25%-35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.