Modeling genetic anencephaly with human brain organoids

点击翻译按钮获取中文摘要

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.