脊髓小脑共济失调3型诱导多能干细胞系的建立和神经分化

Establishment and neural differentiation of spinocerebellar ataxia type 3-induced pluripotent stem cell lines

背景:脊髓小脑共济失调3型(spinocerebellar ataxia type 3,SCA3)是一种典型的遗传相关的神经退行性疾病。建立患者遗传背景的特异的疾病模型有利于研究疾病的发病机制,探讨针对疾病的治疗手段。目的:观察脊髓小脑共济失调3型诱导多能干细胞系的神经分化效率以及CAG拷贝数的稳定性。方法:临床获取脊髓小脑共济失调3型患者皮肤组织,分离培养患者特异的皮肤成纤维细胞,重编程成纤维细胞获得诱导多能干细胞。对患者特异的诱导多能干细胞和正常人的诱导多能干细胞(NHF)和胚胎干细胞(ES-10)进行神经诱导分化,通过流式细胞术比较分化效率,Western Blot检测神经元中ataxin-3的聚集,PCR检测培养过程SCA3/ATXN3基因CAG重复数目。结果与结论:成功获得与成纤维细胞相同遗传背景的诱导多能干细胞,具有与人胚干细胞相似的形态学及多向分化潜能,各细胞系都能定向分化为神经干细胞,重编程前后和诱导神经分化前后的CAG数目无明显改变。来源于脊髓小脑共济失调3型患者的诱导多能干细胞分化为神经干细胞的效率低于正常人诱导多能干细胞(NHF)和胚胎干细胞(ES-10)。这些结果说明通过重编程技术可以成功建立人诱导多能干细胞并将其定向诱导分化为神经干细胞,整个实验过程CAG数目无明显改变,与患者的体细胞相一致。

BACKGROUND：Spinocerebel ar ataxia type 3 （SCA3） is a typical genetic neurodegenerative disease. To establish patient-specific disease models of genetic background contributes to studying the pathogenesis and exploring therapeutic manners. OBJECTIVE：To observe the effectiveness of neural differentiation of induced pluripotent stem cel lines induced by SCA3 and the stability of CAG copy number. METHODS：Skin tissue of SCA3 patient was obtained clinical y, and specific skin flbroblasts were isolated andcultured. Reprogramming fibroblasts could obtain induced pluripotent stem cel s. Patient-specific induced pluripotent stem cel s, normal person induced pluripotent stem cel s （NHF） and embryonic stem cel s （ES-10） were induced to differentiate. Flow cytometry was used to compare the efficiency of differentiation. Western blot assay was utilized to detect ataxin-3 protein expression in neurons. Polymerase chain reaction was applied to measure the CAG repeat number of SCA3/ATXN3 gene. RESULTS AND CONCLUSION：Induced pluripotent stem cel s that had identical genetic background to fibroblasts were successful y obtained, and had similar morphology and multi-directional differentiation potential to human embryonic stem cel s. Each cel line could differentiate into neural stem cel s. The CAG number did not apparently alter before and after reprogramming as wel as induction of neuronal differentiation. The effectiveness of the differentiation of induced pluripotent stem cel s derived from SCA3 into neural stem cel s was lower than that of normal person-derived induced pluripotent stem cel s （NHF） and embryonic stem cel s （ES-10）. These findings demonstrate that reprogramming can successful y establish human induced pluripotent stem cel s, and induced the differentiation of above cel s into neural stem cel s. In the whole process, CAG number did not obviously alter, which was consistent with body cel s of patients.