Institute of Neuroanatomy, Hannover Medical School, 30625, Hannover, Germany.
Cell Tissue Res. 2010 Apr;340(1):29-43. doi: 10.1007/s00441-010-0933-4. Epub 2010 Feb 23.
Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.
神经祖细胞 (NPCs) 具有在再生医学中应用的巨大潜力。为了克服其有限的有丝分裂能力，已经应用了各种永生化策略，使它们能够在体外长期维持和扩增。这些永生化细胞可用于设计和发现用于神经退行性疾病（如帕金森病）的新型基于细胞的治疗方法。我们通过使用 SV40 大 T 抗原 (SV40Tag) 使大鼠腹侧中脑 NPC 永生化。与原代细胞相比，所有细胞克隆的增殖率都提高了两到三倍。为了诱导生成的细胞克隆向多巴胺能分化，同时应用胶质衍生神经营养因子和二丁酰环腺苷单磷酸。然后用多巴胺能谱系标志物的表达、各种细胞群的分化、在海人藻酸存在下的钙成像以及纹状体移植后的免疫组织化学对处理后的细胞进行表征。处理后的细胞显示出形态成熟，并且在海人藻酸存在下的钙成像显示出神经元特性。这些细胞还表达低水平的多巴胺转运体和酪氨酸羟化酶 (TH) mRNA，但未发现 TH 免疫阳性神经元。纹状体移植到神经毒素损伤的大鼠中并未诱导进一步分化。作为替代方法，我们用短发夹 RNA 沉默 SV40Tag，但这不足以触发向多巴胺能神经元分化。尽管如此，β-微管蛋白 III 和神经胶质纤维酸性蛋白染色分别显示出神经元和神经胶质细胞的存在。SV40Tag 细胞适合进行受控的基因修饰，如通过有效的非病毒转染后过表达增强型绿色荧光蛋白所示。
