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Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders
Published on: April 14, 2017
Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19
Wei Niu1,2, Lu Deng1,3, Sandra P Mojica-Perez1
1Department of Neurology, University of Michigan, Ann Arbor, MI, United States.
Insights
Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) in females arises from mosaic PCDH19 expression disrupting cell interactions. This study developed a human cortical organoid model to investigate PCE mechanisms and test therapies.
Area of Science:
- Developmental neuroscience
- Genetics
- Epilepsy research
Background:
- Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a severe neurodevelopmental disorder caused by PCDH19 gene variants.
- PCE primarily affects females due to X-chromosome inactivation leading to mosaic PCDH19 expression.
- Existing models like rodents and iPSCs have limitations in replicating human brain development and mosaicism.
Purpose of the Study:
- To develop a novel in vitro model for mosaic PCDH19 expression in human cortical organoids (hCOs).
- To investigate the role of PCDH19 in human neural development and the pathogenesis of PCE.
- To establish a platform for testing potential precision therapies for PCE.
Main Methods:
- Generated isogenic human embryonic stem cells with wild-type (WT) or knockout (KO) PCDH19 using genome editing.
- Created mosaic hCOs by mixing GFP-labeled WT and RFP-labeled KO cells.
- Analyzed cell sorting, gene expression (PCDH19, N-Cadherin), and neurogenesis in hCOs.
Main Results:
- Mosaic PCE hCOs showed abnormal cell segregation in the ventricular zone (VZ) with distinct WT and KO cell populations.
- PCDH19 KO cells segregated from WT cells even at varying ratios (2:1, 1:2).
- PCE hCOs exhibited altered PCDH19 and N-Cadherin expression and impaired deep layer neurogenesis, unlike control hCOs.
Conclusions:
- PCDH19 is crucial for radial glial organization and early cortical development in humans.
- The mosaic PCE hCO model accurately reflects key aspects of the disease.
- This model provides a valuable tool for understanding PCE mechanisms and developing targeted treatments.
Introduction:
Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation.
Methods:
To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs).
Results:
We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells.
Discussion:
Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.
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