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Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders
Published on: April 14, 2017
Modeling Human Primary Microcephaly With hiPSC-Derived Brain Organoids Carrying CPAP-E1235V Disease-Associated Mutant
Hsiao-Lung An1,2, Hung-Chih Kuo3, Tang K Tang1,2
1Program in Molecular Medicine, National Yang Ming Chiao Tung University and Academia Sinica, Taipei, Taiwan.
Abstract:
The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype-phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.
Insights
Human stem cells with a CPAP mutation create smaller brain organoids, mimicking microcephaly. This model reveals how centrosome dysfunction causes this neurodevelopmental disorder.
Area of Science:
- Cell Biology
- Developmental Neuroscience
- Genetics
Background:
- The centrosome, a key microtubule-organizing center, is crucial for cell division and organization.
- Dysfunction of centrosomal proteins is linked to autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder.
- Existing mouse models fail to fully replicate human-specific MCPH features during brain development.
Purpose of the Study:
- To generate and characterize human induced pluripotent stem cells (hiPSCs) with a specific CPAP mutation (CPAP-E1235V).
- To investigate the phenotypic consequences of this mutation in hiPSCs and derived brain organoids.
- To establish a human-specific model for studying MCPH pathogenesis.
Main Methods:
- CRISPR-Cas9 genome editing to create isogenic hiPSCs with the CPAP-E1235V mutation.
- Analysis of protein recruitment to centrioles in wild-type and mutant hiPSCs.
- Generation and examination of brain organoids derived from both wild-type and mutant hiPSCs.
- Assessment of cellular events, including neuronal differentiation, cell death, and spindle orientation.
Main Results:
- The CPAP-E1235V mutation disrupted centriole elongation by affecting key protein recruitment, leading to short centrioles and long cilia.
- hiPSC-derived brain organoids recapitulated human brain development, including neuronal differentiation and cortical lamination.
- Mutant brain organoids exhibited p53-dependent neuronal cell death, resulting in smaller organoids mimicking the microcephaly phenotype.
- The mutation altered neuronal progenitor cell spindle orientation and induced premature neuronal differentiation.
Conclusions:
- hiPSC-derived brain organoids combined with CRISPR/Cas9 technology provide a robust model for recapitulating MCPH pathology.
- The study elucidates specific cellular mechanisms linking centrosome/centriole dysfunction to microcephaly.
- This model offers new avenues for understanding genotype-phenotype relationships in human neurodevelopmental disorders.
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