Induction of functional photoreceptor phenotype by exogenous Crx expression in mouse retinal stem cells
Induction of functional photoreceptor phenotype by exogenous Crx expression in mouse retinal stem cells
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DOI：
10.1167/iovs.07-0812
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发表时间：
2008-01-01
影响因子：
4.4
通讯作者：
Jones, Stephen E.
中科院分区：
文献类型：
--
作者：
Jomary, Catherine;Jones, Stephen E.
目的。本研究旨在确定转录因子Crx的外源表达是否能促进小鼠视网膜干细胞（RSCs）分化为具有光敏特性的功能性光受体表型的细胞。从小鼠睫状上皮中分离的RSCs经无血清培养后，通过电穿孔法进行基因修饰，表达外源性表位标记的小鼠Crx。干细胞标志物（同源结构域转录因子Pax6、POU转录因子Oct3/4、增殖细胞核抗原[PCNA]）表达的变化；神经元标志物（巢蛋白、神经元特异性III类β -微管蛋白[β III Tub]和神经丝[NF 200]）；以及光感受器特异性标记物（视紫红质[Rho]、环核苷酸门控阳离子通道-3 [CNG3]、蓝锥视蛋白和cGMP磷酸二酯酶[PDE]）；通过免疫细胞化学和Western blot分析来评估分化过程。光导级联活性通过cGMP酶联免疫法测定光诱导的环(c) GMP水解水平来评估。在63%的RSCs中观察到瞬时Crx转基因表达。外源Crx的表达显著降低了干细胞增殖和多能性标志物Pax6、PCNA和Oct3/4的表达。同时，Crx诱导所分析的神经元和光感受器特异性标记物的表达。在表达外源Crx的RSCs中，光诱导的cGMP水解增加，PDE的抑制导致cGMP水平升高。Crx阻止了RSCs的增殖，并诱导它们分化为表达光受体特异性标记的细胞，并表现出可激活的视觉光传导级联的光诱导敏感性特征。本研究表明，Crx可以成功诱导RSCs分化为具有功能性光受体表型的细胞。
PURPOSE. This study was undertaken to determine whether exogenous expression of the transcription factor Crx can promote the differentiation of mouse retinal stem cells (RSCs) into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties.METHODS. RSCs isolated from mouse ciliary epithelium and maintained in serum-free culture were genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6; POU transcription factor Oct3/4; proliferating cell nuclear antigen [PCNA]); of neuronal markers (nestin, neuron-specific class III beta-tubulin [beta III Tub] and neurofilament [NF 200]); and of photoreceptor-specific markers (rhodopsin [Rho], cyclic nucleotide-gated cation channel-3 [CNG3], blue-cone opsin, and cGMP phosphodiesterase [PDE]); were evaluated during differentiation by immunocytochemistry and Western blot analysis. Phototransduction cascade activity was assessed by measuring light-induced hydrolysis of cyclic (c) GMP levels with a cGMP enzyme-linked immunoassay.RESULTS. Transient Crx transgene expression was observed in 63% of RSCs. Expression of stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4, was significantly decreased by exogenous Crx expression. Concomitantly, Crx induced expression of the analyzed neuron- and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in RSCs expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels.CONCLUSIONS. Crx halted proliferation of RSCs and induced them to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. This study demonstrates that Crx can successfully induce RSCs to differentiate into cells with functional photoreceptor phenotypes.