Preserving cellular aspects of aging in patient-specific models of ALS

在患者特有的ALS模型中保留衰老的细胞方面 项目摘要 诱导多能干细胞生物学在人​神经退行性疾病中的应用前景广阔 建模是因为这些细胞可以产生人脑中的任何细胞，一个活的、服从于 实验操作，具有与单个患者完全相同的基因组成，并显示 以前在尸检和临床样本中发现的神经退行性表型。这些 “患者特异性”​体外测试系统使靶向发现、药物筛选和治疗证据成为可能 在患者细胞中进行概念研究的时间比目前可能的要早得多。 尽管有这些独特的优势，保存年龄作为一个关键的致病风险因素目前是一个主要的 这些系统的局限性。这在一定程度上是由于1)细胞中与年龄相关的特征的丧失 恢复到胚胎状态，以及2)分化方案中的缺陷，无法 从胚胎细胞产生成熟的神经元。直接的转录因子介导的重编程也 将患者成纤维细胞转化为诱导运动神经元是另一种选择，被称为“谱系转换”。 体外培养人神经元​的方法。来自我们实验室的新数据，我们的合作者和其他人的展示 通过谱系转换产生的神经元更好地保留了与年龄相关和与疾病相关的缺陷。 在这项为期一年的可行性研究中，我们将比较从 成纤维细胞(fib-MN)，这些细胞是从重新编程的IPSCs定向分化产生的 相同的成纤维细胞样本(IPSC-MNS)。成纤维细胞样本来自GGGGCC患者 ​C9ORF72​基因的六核苷酸重复序列扩展突变，已知会导致一种形式的 肌萎缩侧索硬化症(ALS)和额颞叶痴呆，以及匹配的对照组。我们的第一个假设是 Fib-MN转录本将明显更类似于死后组织和 肌萎缩侧索硬化患者。我们的合作者Verge Genology创造了一种创新的、大数据驱动的ALS基因 使用​公开和专有的基因表达数据进行表达签名，这些数据来自39项与肌萎缩侧索硬化症相关的研究 将在项目中使用。 核质转运缺陷已成为AGE和​C9-​ALS相关的一种表型 不同之处已经确定。​因此，我们的​第二个假设是FIB-MN将具有显著更多的 与IPSC-MNS相比，年龄相关的核质转运缺陷更明显 免疫荧光共聚焦显微镜。总而言之，这两项研究将证明这一原则 FIB-MNS创建了优越的ALS体外测试系统，保留了与衰老有关的重要疾病方面 对IPSC银行和疾病建模领域产生巨大影响的​。AcuraStem Inc.开发人类 用于临床前人类验证其自身和其中枢神经系统疗法的细胞模型和分析 发展伙伴。

Preserving cellular aspects of aging in patient-specific models of ALS Project Summary Induced pluripotent stem cell (iPSC) biology holds great promise for human ​in vitro ​neurodegenerative disease modeling because these cells can give rise to any cell in the human brain, a living “virtual brain” amenable to experimental manipulation, having the exact same genetic makeup as individual patients and displaying neurodegenerative phenotypes previously identified in postmortem and clinical samples. These “patient-specific” ​in vitro testing systems enable target discovery, drug screening and therapeutic proof of concept studies in patient cells much earlier in the translational process than is currently possible. Despite these unique advantages, the preservation of age as a key pathogenic risk factor is presently a major limitation of these systems. This is in part due to 1) the loss of age-related characteristics in cells that are rejuvenated to an embryonic state, and 2) to deficiencies in the differentiation protocols that are unable to produce mature neurons from embryonic cells. The direct, transcription factor mediated reprogramming, also known as “lineage conversion”, of patient fibroblasts into induced motor neurons represents an alternative approach for generating human neurons ​in vitro. New data from our lab, our collaborators’ and others’ show that neurons generated through lineage conversion better retain age-related and disease-associated deficits. In this 1-year feasibility study we will compare motor neurons generated by lineage conversion from fibroblasts (fib-MNs), with those generated by directed differentiation from iPSCs reprogrammed from the same fibroblast samples (iPSC-MNs). The fibroblast samples are from patients having the GGGGCC hexanucleotide repeat expansion mutation in the ​C9ORF72 ​gene, which is known to cause a form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, and matched controls. Our 1st hypothesis is that fib-MN transcriptomes will be significantly more similar to those of post-mortem tissue and those from ALS patients. Our collaborator Verge Genomics has created an innovative, big-data-driven ALS gene expression signature using ​public and proprietary gene expression data from 39 ALS-relevant studies that we will use in the project. Nucleocytoplasmic transport defects have emerged as one phenotype where both age and ​C9-​ALS related differences have been identified. ​Therefore, our ​2nd hypothesis is that fib-MNs will have significantly more pronounced age-related nucleocytoplasmic transport defects than iPSC-MNs as measured by immunofluorescence confocal microscopy. Taken together, these two studies would prove the principle that fib-MNs create superior ALS in-vitro testing systems retaining important, disease-relevant aspects of aging having tremendous impact ​on the iPSC banking and disease modeling fields. AcuraStem Inc. develops human cell models and assays for preclinical human validation of its own CNS therapeutics, as well as those of its development partners.