Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, USA.
J Mol Neurosci. 2013 Oct;51(2):237-48. doi: 10.1007/s12031-012-9930-2. Epub 2012 Dec 9.
Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hampers therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2-induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary cultures of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within the neural lineage, neurons had the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared with control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. These differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.
脊髓小脑性共济失调 2 型（SCA2）是由 12 号染色体上 ATAXN2 基因编码区的三核苷酸重复（CAG）扩展引起的，该扩展产生了一个延长的、有毒的多谷氨酰胺片段，导致浦肯野细胞丢失。目前尚无有效的治疗方法。阻碍治疗发展的主要障碍之一是缺乏理想的疾病模型。在这项研究中，我们生成并表征了 SCA2 诱导的多能干细胞（iPS）细胞系作为体外细胞模型。从 SCA2 患者和健康供体的皮肤外植体原代培养中收获真皮成纤维细胞（FB）。为了重编程，通过逆转录病毒感染将 hOct4、hSox2、hKlf4 和 hc-Myc 转导到第 3 代 FB 中。成功生成了 SCA2 iPS 和对照 iPS 细胞，它们均表现出典型的干细胞生长模式和正常核型。所有 iPS 细胞系均表达干细胞标志物，并在体外分化为三个胚胎生殖层的细胞。在体外神经分化中，SCA2 iPS 细胞在神经玫瑰花结形成中表现出异常，但成功分化为神经干细胞（NSC）和随后的神经细胞。SCA2 和正常 FB 中的 ataxin-2 表达水平相当；然而，SCA2 NSC 中的 ataxin-2 表达水平低于正常 NSC 和 SCA2 FB。在神经谱系中，神经元的 ataxin-2 表达最丰富。神经生长延时分析表明，与对照神经细胞相比，终末分化的 SCA2 神经细胞寿命较短。SCA2 中的扩展 CAG 重复在整个重编程和神经分化过程中保持稳定。总之，我们建立了第一个疾病特异性的人类 SCA2 iPS 细胞系。这些突变 iPS 细胞具有神经分化的潜力。这些携带突变的分化神经细胞对于 SCA2 发病机制和治疗药物开发的研究具有重要价值。
