Alić Ivan, Kosi Nina, Kapuralin Katarina, Gorup Dunja, Gajović Srećko, Pochet Roland, Mitrečić Dinko
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, 10 000 Zagreb, Croatia; Faculty of Veterinary Medicine, University of Zagreb, 10 000 Zagreb, Croatia.
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, 10 000 Zagreb, Croatia.
Neurosci Lett. 2016 Nov 10;634:32-41. doi: 10.1016/j.neulet.2016.10.001. Epub 2016 Oct 3.
To analyse events following transplantation of stem cells in the brain robust tools for tracing stem cells are required. Here we took advantage of the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1 YFP-16), where yellow fluorescent protein (YFP) is under control of the promoter of Thy1 gene. This allows visualising whole neurons, i.e. their cell body, axons and dendrites. In this work fluorescent cells were followed during embryonic development, in vitro differentiation, and after transplantation in the healthy and stroke-affected mouse brain. During embryonic development Thy1-YFP positive cells were first observed on E12.5 and subsequently located in the prosencephalon, rhombencephalon, spinal cord and peripheral nerves. Quantitative analysis by RT-PCR and immunocytochemistry revealed that Thy1-YFP positive cells during embryo development and in vitro differentiation were expressing nestin and SOX2 then MAP2, β3-tubulin and NeuN. Thy1-YFP positive cells isolated from E14.5 represented 21.88±053% (SD) of the cultivated neurons and this remained constant along in vitro differentiation. On the other hand, proportion of Thy1-YFP positive cells reached 50% of neurons in perinatal and one month old mouse brain. Neural stem cells isolated from Thy1 YFP-16 mouse strain transplanted near hippocampus of the healthy and stroke-affected brain were distinguishable by YFP fluorescence. They differentiated into mature neurons and were detectable even 14 weeks after transplantation, the end point of our experiment. In conclusion, stem cells originating from Thy1 YFP-16 mice represent an outstanding tool to monitor neurogenesis enabling morphological analyses of new neurons and their projections, in particular after transplantation in the brain.
为了分析脑内干细胞移植后的事件，需要强大的工具来追踪干细胞。在这里，我们利用了小鼠品系B6.Cg-Tg(Thy1-YFP)16Jrs/J（Thy1 YFP-16），其中黄色荧光蛋白（YFP）受Thy1基因启动子的控制。这使得可以可视化整个神经元，即它们的细胞体、轴突和树突。在这项工作中，在胚胎发育、体外分化过程中以及在健康和中风影响的小鼠脑内移植后，对荧光细胞进行了追踪。在胚胎发育过程中，Thy1-YFP阳性细胞首先在E12.5被观察到，随后位于前脑、后脑、脊髓和外周神经中。通过RT-PCR和免疫细胞化学进行的定量分析表明，胚胎发育和体外分化过程中的Thy1-YFP阳性细胞表达巢蛋白和SOX2，然后表达MAP2、β3-微管蛋白和NeuN。从E14.5分离的Thy1-YFP阳性细胞占培养神经元的21.88±053%（标准差），并且在体外分化过程中保持恒定。另一方面，在围产期和1月龄小鼠脑中，Thy1-YFP阳性细胞的比例达到神经元的50%。从Thy1 YFP-16小鼠品系分离的神经干细胞移植到健康和中风影响的脑的海马附近，通过YFP荧光可以区分。它们分化为成熟神经元，甚至在移植后14周（我们实验的终点）仍可检测到。总之，源自Thy1 YFP-16小鼠的干细胞是监测神经发生的出色工具，能够对新神经元及其投射进行形态学分析，特别是在脑内移植后。
