Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro.
Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro.
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DOI：
10.1007/s12031-012-9930-2
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发表时间：
2013-10
期刊：
影响因子：
--
通讯作者：
Ashizawa T
中科院分区：
文献类型：
--
作者：
Xia G;Santostefano K;Hamazaki T;Liu J;Subramony SH;Terada N;Ashizawa T
脊髓小脑性共济失调2型（SCA 2）是由12号染色体上ATAXN2基因编码区的三核苷酸重复（CAG）扩增引起的，其产生延长的毒性聚谷氨酰胺束，导致浦肯野细胞丢失。目前没有有效的治疗方法。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，我们已经产生并表征了SCA2诱导的多能干细胞（iPS）细胞系作为体外细胞模型。从SCA 2受试者和健康受试者的皮肤外植体的原代培养物中收获真皮成纤维细胞（FB）。对于重编程，通过逆转录病毒感染将hOct4、hSox2、hKlf4和hc-Myc转导至第3代FB。SCA2 iPS和对照iPS细胞均成功产生，并显示出具有正常核型的典型干细胞生长模式。所有iPS细胞系均表达干细胞标志物，并在体外分化为来自三个胚胎胚层的细胞。在体外神经分化时，SCA2 iPS细胞在神经花结形成中显示出异常，但成功地分化为神经干细胞（NSC）和随后的神经细胞。SCA2和正常FB显示出相当水平的共济失调蛋白-2表达;而SCA2 NSC显示出比正常NSC和SCA2 FB更少的共济失调蛋白-2表达。在神经谱系中，神经元具有最丰富的ataxin-2表达。时间推移神经生长测定表明，终末分化的SCA2神经细胞与对照神经细胞相比寿命短。SCA2扩增的CAG重复序列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人SCA2 iPS细胞系。这些突变的iPS细胞具有神经分化的潜力。携带突变的分化的神经细胞对于SCA2发病机制的研究和治疗药物的开发是非常宝贵的。
Spinocerebellar ataxia type 2 (SCA 2) 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 hamper 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 culture 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 neural lineage, neurons have the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared to 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. The differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.
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