Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F. 04510, México.
Cell Transplant. 2009;18(10):1171-81. doi: 10.3727/096368909X12483162197123. Epub 2009 Aug 5.
Embryonic stem (ES) cells can be induced to differentiate into motor neurons (MN). Animal models resembling MN degeneration and paralysis observed in familial amyotrophic lateral sclerosis (ALS) have been previously reported. In this work, we aimed to investigate whether transplanted MN could prevent motor deterioration in transgenic rats expressing a mutant form of human superoxide dismutase 1 (hSOD1(G93A)) associated with inherited ALS. Mouse ES cells were differentiated to neurons that express green fluorescent protein (GFP) under the promoter of the MN-specific gene hb9, as well as molecular markers indicative of MN identity. Cells were grafted into the lumbar spinal cord of adult wild-type (WT) or hSOD1(G93A) rats at 10 weeks of age, when transgenic animals are presymptomatic. Grafted cells with MN phenotype can survive for at least 1 week in hSOD1(G93A) animals. To quantitatively evaluate motor performance of WT and transgenic rats, we carried out weekly rotarod tests starting when the animals were 14 weeks old. Sham and grafted WT animals showed no decline in their ability to sustain themselves on the rotating rod. In contrast, sham hSOD1(G93A) rats decreased in motor performance from week 16 onwards, reaching paralysis by week 19 of age. In grafted transgenic animals, there was a significant improvement in rotarod competence at weeks 16 and 17 when compared to sham hSOD1(G93A). However, in the following weeks, transplanted hSOD1(G93A) rats showed motor deterioration and eventually exhibited paralysis by week 19. At end-stage, we found only a few endogenous MN in sham and grafted hSOD1(G93A) rats by cresyl violet staining; no choline acetyl transferase-positive nor GFP-positive MN were present in grafted transgenic subjects. In contrast, WT rats analyzed at the same age possessed grafted GFP-positive MN in their spinal cords. These results strongly suggest that the transgenic hSOD1(G93A) environment is detrimental to grafted MN in the long term.
胚胎干细胞（ES 细胞）可被诱导分化为运动神经元（MN）。先前已有报道称，已建立了类似于家族性肌萎缩侧索硬化症（ALS）中观察到的 MN 变性和瘫痪的动物模型。在这项工作中，我们旨在研究是否移植 MN 可以预防表达与遗传性 ALS 相关的人超氧化物歧化酶 1（hSOD1（G93A））突变体的转基因大鼠的运动恶化。将小鼠 ES 细胞分化为在 MN 特异性基因 hb9 的启动子下表达绿色荧光蛋白（GFP）的神经元，以及指示 MN 特征的分子标记物。在转基因动物出现症状前的 10 周龄时，将细胞移植到成年野生型（WT）或 hSOD1（G93A）大鼠的腰脊髓中。具有 MN 表型的移植细胞可以在 hSOD1（G93A）动物中至少存活 1 周。为了定量评估 WT 和转基因大鼠的运动性能，我们从动物 14 周龄开始每周进行旋转棒测试。假手术和移植的 WT 动物在旋转棒上的维持能力没有下降。相比之下，假手术 hSOD1（G93A）大鼠从第 16 周开始运动性能下降，到第 19 周时达到瘫痪。在移植的转基因动物中，与假手术 hSOD1（G93A）相比，第 16 和 17 周的旋转棒能力有显著改善。然而，在接下来的几周中，移植的 hSOD1（G93A）大鼠出现运动恶化，最终在第 19 周时出现瘫痪。在终末期，通过 Cresyl Violet 染色，我们仅在假手术和移植的 hSOD1（G93A）大鼠中发现了少量内源性 MN；在移植的转基因受体内未发现胆碱乙酰转移酶阳性或 GFP 阳性 MN。相比之下，在相同年龄分析的 WT 大鼠在其脊髓中具有移植的 GFP 阳性 MN。这些结果强烈表明，在长期内，转基因 hSOD1（G93A）环境对移植的 MN 有害。
