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Immortalized striatal precursor neurons from Huntington’s disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics
Human Molecular Genetics ( IF 3.1 ) Pub Date : 2021-07-16 , DOI: 10.1093/hmg/ddab200
Sergey S Akimov 1 , Mali Jiang 1 , Amanda J Kedaigle 2 , Nicolas Arbez 1 , Leonard O Marque 1 , Chelsy R Eddings 1 , Paul T Ranum 3 , Emma Whelan 1 , Anthony Tang 1 , Ronald Wang 1 , Lauren R DeVine 4 , Conover C Talbot 5 , Robert N Cole 4 , Tamara Ratovitski 1 , Beverly L Davidson 3, 6 , Ernest Fraenkel 2 , Christopher A Ross 1, 7
Affiliation  

We have previously established induced pluripotent stem cell (iPSC) models of Huntington’s disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.

中文翻译:


来自亨廷顿病患者的 iPS 细胞的永生化纹状体前体神经元作为实验治疗靶点识别和筛选的平台



我们之前建立了亨廷顿病 (HD) 的诱导多能干细胞 (iPSC) 模型,证明了 CAG 重复扩增依赖性细胞生物学变化和毒性。然而,当前的分化方案繁琐且耗时,使得制备大量细胞用于生化或筛选测定变得困难。在这里,我们报告了 HD iPSC 中具有正常 (33) 和扩展 (180) CAG 重复的永生化纹状体前体神经元 (ISPN) 的生成,分化为类似于中型多棘神经元 (MSN) 的表型,作为更多原理的证明易于处理的患者来源的细胞模型。为了实现永生化,我们使用了端粒酶 hTERT 的酶促成分和 c-Myc 的条件表达的共表达。正如免疫细胞化学标准所证明的,ISPN 可以作为稳定的贴壁细胞系繁殖,并在 2 周内快速分化为高度均质的 MSN 样培养物。分化的 ISPN 概括了亲本 iPSC 模型的主要 HD 相关表型,包括脑源性神经营养因子 (BDNF) 戒断诱导的细胞死亡,这种死亡可以通过先前在亲本 iPSC 模型中验证的小分子来挽救。蛋白质组和 RNA-seq 分析通过主成分分析证明了 HD 样品与对照样品的分离。我们确定了几个网络、通路和上游调节器,还在 HD iPSC、其他 HD 模型和 HD 患者样本中发现了改变。 HD ISPN 系可用于研究 HD 相关的细胞发病机制,并可用作 HD 靶点识别和筛选实验疗法的平台。 所描述的从分化的患者来源的 iPSC 生成 ISPN 的方法可以应用于更大的 HD 细胞系等位基因系列,以及其他遗传疾病的类似模型。
更新日期:2021-07-16
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