Jankowiak Wanda, Kruszewski Katharina, Flachsbarth Kai, Skevas Christos, Richard Gisbert, Rüther Klaus, Braulke Thomas, Bartsch Udo
Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Department of Ophthalmology, Sankt Gertrauden-Krankenhaus, Berlin, Germany.
PLoS One. 2015 May 20;10(5):e0127204. doi: 10.1371/journal.pone.0127204. eCollection 2015.
A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the nclf mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old nclf mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.
持续眼内给药神经营养因子是旨在为目前无法治疗的退行性视网膜疾病建立治疗方法的策略之一。在本研究中,我们分析了基于神经干细胞(NS)持续眼内递送睫状神经营养因子(CNTF)对nclf小鼠光感受器细胞的神经保护作用,nclf小鼠是神经退行性溶酶体贮积症变异型晚发性婴儿神经元蜡样脂褐质沉积症(vLINCL)的动物模型。为此,我们用一种多顺反子慢病毒载体对贴壁培养的NS细胞进行基因改造,该载体编码可分泌的CNTF变体以及Venus报告基因(CNTF-NS细胞)。用于对照实验的NS细胞(对照-NS细胞)用编码报告基因tdTomato的载体进行改造。使用荧光激活细胞分选建立克隆性CNTF-NS和对照-NS细胞系,并在视网膜变性开始时将其玻璃体内移植到14日龄的nclf小鼠中。移植的细胞优先分化为星形胶质细胞,这些细胞附着在晶状体后侧和视网膜玻璃体一侧,并至少在六周内稳定表达转基因,这是分析的最晚移植后时间点。未观察到供体细胞整合到宿主视网膜中、移植细胞的持续增殖或供体细胞对宿主眼睛形态的不良影响。细胞移植后两周、四周和六周对宿主视网膜的定量分析显示,移植了CNTF-NS细胞的眼睛中的光感受器细胞明显多于移植了对照-NS细胞 的眼睛。这首次证明了在神经元蜡样脂褐质沉积症动物模型中,持续眼内给药神经营养因子可减轻视网膜变性。