Modeling genetic anencephaly with human brain organoids

用人脑类器官模拟遗传性无脑畸形

• 批准号: 10370452
• 负责人: Andrew M Tidball
• 金额: $ 13.09万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-07 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10370452
• 关键词: Amniocentesis; Anencephaly; Apical; Area; Biological Models; Blood; Blood specimen; Brain; CELSR1 gene; CRISPR/Cas technology; Cell Line; Cells; Cephalic; Chemicals; Chorionic Villi Sampling; Chorionic villi; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Confocal Microscopy; Congenital Abnormality; Congenital neurologic anomalies; DNA; DNA Sequence Alteration; Development; Disease; Engineering; Enrollment; Environmental Exposure; Etiology; Failure; Family; Fertility; Fetal Death; Fetus; Fibroblasts; Fluorescence Microscopy; Folic Acid; Frameshift Mutation; Future; Gene Mutation; Genes; Genetic; Genetic Models; Genome; Genome Stability; Goals; Heterogeneity; Human; Human Genetics; Image; Informed Consent; Investigation; Knock-out; Label; Lead; Light; Maternal-fetal medicine; Measurement; Measures; Methods; Modeling; Molecular Conformation; Mononuclear; Mutagenesis; Mutation; Neural Tube Closure; Neural Tube Defects; Neural tube; Newborn Infant; Organoids; Parents; Pathway interactions; Patients; Phase; Phenotype; Pregnancy; Prophylactic treatment; Proteins; Publishing; Quantitative Reverse Transcriptase PCR; Recommendation; Reproducibility; Research Personnel; Severities; Surface; Techniques; Technology; Teratogens; Umbilical Cord Blood; Validation; Variant; causal variant; cell bank; clinical practice; constriction; cranium; de novo mutation; exome; exome sequencing; expectation; fetal; folic acid supplementation; foot; gene correction; genetic variant; human model; in utero; induced pluripotent stem cell; inhibitor; member; mouse model; neonate; neurodevelopment; neuroteratogen; novel; planar cell polarity; pluripotency; proband; screening

Abstract Neural tube defects (NTDs) are common malformations of the nervous system that occur during pregnancy. Anencephaly is one of the most dramatic and devastating NTDs characterized by failure of the brain and skull to close during neurulation. It is always fatal. Recent trio exome sequencing studies have identified de novo genetic mutations (DNMs) in anencephalic fetuses, and some of these mutations have been in genes associated with anencephaly in genetic mouse models. For these reasons, DNMs are beginning to be considered an important factor in the etiology of anencephaly and other NTDs. However, current model systems do not easily allow for directly validating that these variants lead to NTDs. Having a human-specific model of early neurodevelopment would allow for these types of conformational studies and further mechanistic investigations. Attempts to utilize human brain organoid technology to this end are limited due to structural heterogeneity and intra-organoid variability. Our recent development of reproducible self-organizing single rosette spheroids (SOSRS) from human induced pluripotent stem cells has allowed us to treat SOSRS with two known neuroteratogens and observe distinct structural changes consistent with NTDs. The goal in Aim 1 is to generate a model of anencephaly by knocking out genes known to lead to anencephaly (SHROOM3 and CELSR1) in SOSRS and characterize structural signatures indicative of NTDs. In Aim 2, we will generate fetal-specific models. To this end, we will enroll families with anencephalic fetuses to obtain blood samples, cord blood, and/or amniocentesis. Blood DNA will be used to perform trio exome sequencing to identify DNMs. Cells from the cord blood or amniocentesis from anencephalic fetuses with likely causative DNMs will be reprogrammed into iPSCs. The potentially causative mutations will also be corrected by CRISPR/Cas9 to generate isogenic controls. Finally, SOSRS from these models will be generated and measured for phenotypes identified in the proof-of-principle anencephalic models. Our study will likely shed light on the mechanisms of human anencephaly and allow for screening novel DNMs for NTD causality, thus, greatly expanding our understanding of human NTD genetics. Furthermore, our collaboration of basic researchers with clinicians specializing in genetics of maternal and fetal medicine will provide a framework for rapid modeling of clinical cases, potentially resulting in altered clinical practices including fertility recommendations and the utility of prophylactic treatments, such as folate supplementation.

摘要 神经管缺陷（NTD）是妊娠期间发生的神经系统常见畸形。 无脑畸形是一种最具戏剧性和破坏性的神经管畸形，其特征是大脑和颅骨的功能衰竭 在神经形成期关闭它总是致命的。最近的三个外显子组测序研究已经确定了从头 基因突变（DNMs）在无脑胎儿，其中一些突变已在基因 与遗传小鼠模型中的无脑畸形相关。由于这些原因，DNM开始成为 被认为是无脑畸形和其他NTD病因学的重要因素。然而，目前的模式 系统不容易允许直接验证这些变体导致NTD。具有人类特有的 早期神经发育的模型将允许这些类型的构象研究，并进一步 机械研究。为此目的利用人脑类器官技术的尝试受到限制， 结构异质性和类器官内变异性。我们最近发展的可再生自组织 来自人类诱导多能干细胞的单玫瑰花结球体（SOSRS）使我们能够治疗SOSRS 用两种已知的神经致畸剂，观察到与NTD一致的明显结构变化。年的目标 目的1是通过敲除已知导致无脑畸形的基因来产生无脑畸形模型 （SHROOM 3和CELSR 1）在SOSRS中并表征指示NTD的结构特征。在目标2中， 将生成胎儿特定的模型。为此，我们将招募无脑畸形胎儿的家庭， 样本、脐带血和/或脐带穿刺术。血液DNA将用于进行三外显子组测序， 识别DNM。来自脐带血或无脑畸形胎儿的脐带穿刺的细胞， DNM将被重新编程为iPSC。潜在的致病突变也将被纠正， CRISPR/Cas9以产生同基因对照。最后，将从这些模型中生成SOSRS， 测量在原理证明无脑模型中鉴定的表型。我们的研究很可能会 阐明人类无脑畸形的机制，并允许筛选NTD因果关系的新DNM，因此， 极大地扩展了我们对人类NTD遗传学的理解。此外，我们的合作基础 研究人员与临床医生专门从事遗传学的孕产妇和胎儿医学将提供一个框架， 临床病例的快速建模，可能导致临床实践（包括生育）的改变 建议和预防性治疗的效用，如叶酸补充剂。