Clinical Neurosciences Division, University of Southampton, South Laboratory and Pathology Block, Southampton General Hospital, Southampton, United Kingdom.
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1181-9. doi: 10.1167/iovs.09-4385. Epub 2009 Oct 22.
This study compared the effect of the transcription factor Crx (cone, rod homeobox) on the differentiation of human adult corneal (hCSC) and retinal (hRSC) stem cells into functional photoreceptors.
Stem cells isolated from postmortem human corneas and retinal ciliary bodies were maintained in serum-free culture and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Expression of stem cell markers (Pax6, Oct3/4, and proliferating cell nuclear antigen [PCNA]), neuronal markers (nestin, neuron-specific class III beta-tubulin, Map2 a/b, and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, and beta-6-PDE) was evaluated by immunocytochemistry. A cGMP enzyme-linked immunoassay was used to assess phototransduction cascade activity by measurement of light-induced hydrolysis of cGMP.
Expression of the stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4 was decreased by exogenous Crx expression in both hCSCs and hRSCs. Correspondingly, the expression of the mature neuronal markers Map2 a/b and neurofilament was increased. Both hCSCs and hRSCs displayed photoreceptor-specific immunolabeling. However, light-activated GMP hydrolysis was observed only in hRSCs after exogenous expression of Crx.
The present study extends previous findings that exogenous Crx expression can promote differentiation of human retina-derived stem cells into light-sensitive photoreceptor phenotypes. Although Crx can induce human cornea-derived stem cells to express photoreceptor-specific proteins, it does not seem to be sufficient to direct their differentiation into functional photoreceptors. Nevertheless, this study demonstrates that genetic modification of adult human retinal stem cells can cause differentiation into light-sensitive photoreceptor phenotypes.
本研究比较了转录因子 Crx（视锥、视杆同源盒）对人眼角膜（hCSC）和视网膜（hRSC）干细胞分化为功能性光感受器的影响。
从死后人眼角膜和视网膜睫状体分离的干细胞在无血清培养中维持，并通过电穿孔进行基因修饰以表达外源性表位标记的鼠 Crx。通过免疫细胞化学评估干细胞标志物（Pax6、Oct3/4 和增殖细胞核抗原 [PCNA]）、神经元标志物（巢蛋白、神经元特异性 III 类 β-微管蛋白、Map2 a/b 和神经丝）和光感受器特异性标志物（视紫红质、环核苷酸门控阳离子通道-3、蓝锥光感受器和β-6-PDE）的表达。通过测量 cGMP 光诱导水解来评估 cGMP 酶联免疫吸附试验来评估光转导级联活性。
在 hCSC 和 hRSC 中外源 Crx 表达降低了增殖和多能性干细胞标志物 Pax6、PCNA 和 Oct3/4 的表达。相应地，成熟神经元标志物 Map2 a/b 和神经丝的表达增加。hCSC 和 hRSC 均显示光感受器特异性免疫标记。然而，只有在 hRSC 中外源表达 Crx 后才观察到光激活的 GMP 水解。
本研究扩展了先前的发现，即外源 Crx 表达可以促进人视网膜衍生干细胞分化为对光敏感的光感受器表型。尽管 Crx 可以诱导人眼角膜来源的干细胞表达光感受器特异性蛋白，但似乎不足以将其分化为功能性光感受器。然而，本研究表明，成年人类视网膜干细胞的基因修饰可以导致分化为对光敏感的光感受器表型。
