Qiu Guanting, Seiler Magdalene J, Thomas Biju B, Wu Kebin, Radosevich Michael, Sadda SriniVas R
Department of Ophthalmology, Doheny Retina Institute, Keck School of Medicine, at the University of Southern California, 1450 Pablo St - DEI 3610, Los Angeles, CA, 90033-3699, USA.
Exp Eye Res. 2007 Jun;84(6):1047-59. doi: 10.1016/j.exer.2007.01.014. Epub 2007 Feb 2.
The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.
本研究的目的是在体外扩增的视网膜祖细胞（RPCs）中，对神经巢蛋白（一种神经外胚层干细胞和反应性神经胶质细胞标志物）与各种成熟视网膜细胞标志物的共表达进行表征，随后进行体外诱导或视网膜下移植。从胚胎第17天（E17）大鼠视网膜分离得到的RPCs在无血清限定培养基中扩增，并用全反式维甲酸（RA）诱导分化。诱导后，细胞用神经巢蛋白与视网膜神经元和神经胶质细胞标志物进行联合染色。诱导前后收集培养细胞进行定量RT-PCR基因表达分析。在第二个系列实验中，将第2代RPCs在出生后第28天移植到S334ter-3视网膜变性大鼠的视网膜下间隙。1-4周后，对移植部位的切片进行神经巢蛋白和各种视网膜特异性神经元标志物的双重免疫染色。用RA处理的培养RPCs表现出神经巢蛋白与各种视网膜特异性标志物的共表达，包括蛋白激酶Cα（PKC）、神经丝200（NF200）、细胞视黄醛结合蛋白（CRALBP）和视紫红质。RA诱导后，定量RT-PCR分析显示神经巢蛋白、PAX-6、thy1.1和PKCα下调，视紫红质、胶质纤维酸性蛋白（GFAP）和CrX上调。在体内RPC移植中，除了宿主视网膜中一些神经巢蛋白免疫反应性与GFAP阳性细胞重叠外，未观察到神经巢蛋白与任何视网膜特异性神经元标志物的共表达。神经巢蛋白作为一种独特的神经干细胞/祖细胞标志物的作用应重新考虑。RPC成熟过程中神经巢蛋白的表达在体外和体内似乎有所不同。
