Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines
Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the <i>OPA1</i> gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion...
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2025-01-01
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| author | Katherine A. Pohl Xiangmei Zhang Johnny Jeonghyun Ji Linsey Stiles Alfredo A. Sadun Xian-Jie Yang |
| author_facet | Katherine A. Pohl Xiangmei Zhang Johnny Jeonghyun Ji Linsey Stiles Alfredo A. Sadun Xian-Jie Yang |
| author_sort | Katherine A. Pohl |
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| description | Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the <i>OPA1</i> gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although <i>OPA1</i> is broadly expressed, human <i>OPA1</i> mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate <i>OPA1</i> mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both <i>OPA1</i> homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the <i>OPA1</i> mutation in a DOA patient’s iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, <i>OPA1</i> mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing <i>OPA1</i>-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration. |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-133b971757da4f33bcca29e3f8f4d7592025-01-24T13:26:49ZengMDPI AGCells2073-44092025-01-0114213710.3390/cells14020137Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell LinesKatherine A. Pohl0Xiangmei Zhang1Johnny Jeonghyun Ji2Linsey Stiles3Alfredo A. Sadun4Xian-Jie Yang5Jules Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USAJules Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USAJules Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADepartment of Molecular and Medical Pharmacology, Davide Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADoheny Eye Center, Department of Ophthalmology, University of California, Los Angeles, CA 91103, USAJules Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the <i>OPA1</i> gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although <i>OPA1</i> is broadly expressed, human <i>OPA1</i> mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate <i>OPA1</i> mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both <i>OPA1</i> homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the <i>OPA1</i> mutation in a DOA patient’s iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, <i>OPA1</i> mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing <i>OPA1</i>-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration.https://www.mdpi.com/2073-4409/14/2/137<i>OPA1</i> genedominant optic atrophyCRISPR-Cas9 editingisogenic human pluripotent stem cell linesmitochondria |
| spellingShingle | Katherine A. Pohl Xiangmei Zhang Johnny Jeonghyun Ji Linsey Stiles Alfredo A. Sadun Xian-Jie Yang Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines Cells <i>OPA1</i> gene dominant optic atrophy CRISPR-Cas9 editing isogenic human pluripotent stem cell lines mitochondria |
| title | Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines |
| title_full | Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines |
| title_fullStr | Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines |
| title_full_unstemmed | Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines |
| title_short | Derivation and Characterization of Isogenic <i>OPA1</i> Mutant and Control Human Pluripotent Stem Cell Lines |
| title_sort | derivation and characterization of isogenic i opa1 i mutant and control human pluripotent stem cell lines |
| topic | <i>OPA1</i> gene dominant optic atrophy CRISPR-Cas9 editing isogenic human pluripotent stem cell lines mitochondria |
| url | https://www.mdpi.com/2073-4409/14/2/137 |
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