Cesnulevicius Konstantin, Timmer Marco, Wesemann Maike, Thomas Tobias, Barkhausen Tanja, Grothe Claudia
Department of Neuroanatomy, Center for Systems Neuroscience, Hannover, Germany.
Stem Cells. 2006 Dec;24(12):2776-91. doi: 10.1634/stemcells.2006-0176. Epub 2006 Aug 10.
Neuronal progenitor cells (NPCs) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, such as Parkinson disease. However, survival of transplanted cells is, as yet, limited, and the identification of grafted cells in situ remains difficult. The use of NPCs could be more effective with regard to a better survival and maturation when transfected with one or more neurotrophic factors. Therefore, we investigated the possibility of transfecting mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters enhanced green fluorescence protein (EGFP) and red fluorescent protein (DsRed). Different techniques for transfection were compared, and the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure; 6 hours after transfection, viability was approximately 40%, and the transfected cells differentiated into, for example, tyrosine hydroxylase-positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. To provide the progenitor cells with a neurotrophic factor, different isoforms of fibroblast growth factor-2 were introduced. To follow the behavior of the transfected cells in vitro, functional tests such as the cell viability assay (water-soluble tetrazolium salt assay [WST-1]) and the cell proliferation assay (5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay) were performed. In addition, these transfected NPCs were viable after transplantation, expressed tyrosine hydroxylase in vivo, and could easily be detected within the host striatum because of their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases.
神经元祖细胞（NPCs）在神经退行性疾病（如帕金森病）的潜在再生治疗策略中发挥着重要作用。然而，移植细胞的存活率目前仍然有限，并且在原位识别移植细胞仍然很困难。当用一种或多种神经营养因子转染时，NPCs的使用在更好的存活和成熟方面可能更有效。因此，我们研究了用携带神经营养因子或表达报告基因增强型绿色荧光蛋白（EGFP）和红色荧光蛋白（DsRed）的不同构建体转染中脑神经元祖细胞的可能性。比较了不同的转染技术，通过核转染实现了高达47%的最高转染率。中脑神经元祖细胞在转染过程中存活下来；转染后6小时，存活率约为40%，并且转染的细胞分化为例如酪氨酸羟化酶阳性神经元。在转染细胞组中，发现了许多祖细胞和几个神经元。为了给祖细胞提供一种神经营养因子，引入了成纤维细胞生长因子-2的不同异构体。为了在体外追踪转染细胞的行为，进行了功能测试，如细胞活力测定（水溶性四唑盐测定法[WST-1]）和细胞增殖测定（5-溴-2'-脱氧尿苷-酶联免疫吸附测定）。此外，这些转染的NPCs在移植后是存活的，在体内表达酪氨酸羟化酶，并且由于其EGFP表达，在宿主纹状体内很容易被检测到。这项研究表明，神经祖细胞的基因修饰可为神经退行性疾病的新治疗概念提供有吸引力的前景。
