Using iPSCs and cerebral organoids to dissect the pathophysiology of brain growth disorders
Filomena Pirozzi1, Gaia Ruggeri1, Matthew Berkseth1, Anthony Wynshaw-Boris2, William B. Dobyns1,2, Ghayda M. Mirzaa1,2
1) Center for integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA
2) Department of Genetics and Genome Sciences, Case Western Reserve university, Cleveland, OH
3) Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA
Microcephaly (MIC) and Megalencephaly (MEG) are neurodevelopmental disorders at the opposite ends of the brain growth spectrum, with severe MIC defined as occipitofrontal circumference (OFC) at least 3 standard deviation (SD) below the mean, and MEG presenting with OFC > 3SD above the mean. MIC and MEG are characterized by variable brain and cortical abnormalities. This spectrum is associated with significant pediatric morbidity and mortality including epilepsy, autism and intellectual disability. In this talk, I will present two examples of how our team has used iPSCs and cerebral organoids to model in vitro these two phenotypes, with the aim to identify the molecular cascade of events and, possibly, new drug targets. As example of MIC, we focused on LIG4 Syndrome, a rare autosomal disorder leading to deficiency of the DNA repair pathway Non-Homologous End Joining (NHEJ). In addition, we selected the PI3K-MTOR pathway as example of MEG, as these genes have been identified to cause Focal Cortical Displasya (FCD), the most common cause of pediatric epilepsy. We generated induced pluripotent stem cells (iPSCs) carrying the common LIG4R278H, PIK3CAH1047R or MTORT1977I mutations. We differentiated the obtained iPSCs into Neuronal Progenitors (NPCs), cortical neurons, and cerebral organoids and performed functional assays including population doubling time, senescence, analysis of proliferation and apoptosis. We were able to recapitulate the microcephaly and megalencephaly phenotypes in vitro, identifying different molecular events leading to the pathogenesis of these phenotypes for each gene. Interestingly, despite sharing similar clinical features and being nodes of the same pathways, we revealed both overlapping and exclusive cellular phenotypes in PIK3CA and MTOR mutant cell lines, indicating that classifying MEG based on the pathway might not be enough to discriminate among different pathogenesis. These results, together with previous work from other labs, provide proof-of-concept that iPSC and cerebral organoids are ideal tools to dissect the pathophysiology of brain growth disorders in humans. Our future directions is to use these tools in order to perform high-throughput drug screening in order to identify novel therapeutic candidates.
Filomena Pirozzi, Ph.D., pursued her studies between Italy, Belgium and USA. After her Ph.D. in Molecular Genetics (Catholic University of Rome, Italy), she perfectioned her skills on induced pluripotent stem cells (iPSCs) and human neuronal models during her postdoctoral training at KU Leuven first and at Case Western Reserve University later. Her studies started with a focus on intellectual Disability and fragile X Syndrome, but expanded to neurodevelopmental disorders. Currently, Dr. Pirozzi focuses on generating neuronal models for neurodevelopment disorders at Seattle Children’s Research Institute, including microcephaly and macrocephaly (MIC/MAC). She generates induced pluripotent stem cells from patients’ cells in order to study the molecular mechanisms leading to these two phenotypes. Her aims are to (1) identify genetic causes for primary and syndromic Mic/Mac (2) better define their neuronal pathophysiology using two-dimensional and three-dimensional in vitro models (3) identify candidate targets for drug screening and personalized treatments.