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CRISPR/Cas9-Induced Loss of Keap1 Enhances Anti-oxidation in Rat Adipose-Derived Mesenchymal Stem Cells
Yiling Hu1, Shubao Liu1, Bing-Mei Zhu1
1Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, China.
Abstract:
Stem cells have become a powerful tool in the treatment of many diseases owing to their regenerative ability and rapid promotion of development in regenerative medicine such as in traumatic brain injury. However, the high level of oxidant micro-environment in lesion region leads to more than 99% cells into death. In this study, we used genetic methods to edit Keap1 gene in mesenchymal stem cells, we and observed their antioxidative ability. First, we disturbed the start codon and the 376th amino acid codon of Keap1 in adipose-derived mesenchymal stem cells (Ad-MSCs) with CRISPR/Cas9, respectively, to release Nrf2 from the binding of Keap1. As a result, Nrf2 was activated and localized into nuclei and regulated cellular anti-oxidation. We observed that the cells lacking Keap1 ATG codon showed obvious nuclear localization of Nrf2. Besides lower expression of Bax-1 and lower content of malondialdehyde (MDA) were detected after H2O2 treatment, we also found higher expression of Bcl-2 in Keap1 ATG codon knock-out cells, whereas a higher expression of PCNA was observed only in the Keap1 376th codon-edited cells, whose Bax-1 expression was lower than that in the control cells. Our study revealed that loss of Keap1 resulted in anti-oxidative ability in Ad-MSCs, suggesting that our strategy can hopefully increase the viability of mesenchymal stem cells after grafting. This study is also a frontier exploration to the application of CRISPR/Cas9 in Ad-MSCs.
Insights
Genetic editing of the Keap1 gene in mesenchymal stem cells enhances their antioxidant capacity. This approach, using CRISPR/Cas9 technology, improves cell survival in oxidative environments, crucial for regenerative medicine applications.
Area of Science:
- Regenerative Medicine
- Molecular Biology
- Cell Biology
Background:
- Mesenchymal stem cells (MSCs) are vital for regenerative medicine due to their repair capabilities.
- Oxidative stress in injured tissues, such as traumatic brain injury, causes high MSC death rates (>99%).
- Enhancing MSC antioxidant defense is critical for improving therapeutic outcomes.
Purpose of the Study:
- To investigate the effect of Keap1 gene editing on the antioxidant capacity of adipose-derived mesenchymal stem cells (Ad-MSCs).
- To assess the potential of CRISPR/Cas9 technology for enhancing MSC viability in oxidative environments.
- To explore the role of Keap1 in regulating Nrf2 activation and downstream antioxidant responses in Ad-MSCs.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to disrupt the start codon (ATG) and the 376th amino acid codon of the Keap1 gene in Ad-MSCs.
- Assessed Nrf2 nuclear localization as an indicator of its activation.
- Quantified the expression of antioxidant and apoptosis-related genes (Bax-1, Bcl-2, PCNA) and malondialdehyde (MDA) levels following hydrogen peroxide (H2O2) treatment.
Main Results:
- Keap1 gene editing successfully released Nrf2, leading to its nuclear localization and activation of cellular antioxidant pathways.
- Ad-MSCs with Keap1 ATG codon knockout exhibited enhanced Nrf2 nuclear localization.
- These edited cells showed reduced Bax-1 expression, lower MDA content, and increased Bcl-2 expression after H2O2 treatment, indicating improved oxidative stress resistance.
- Keap1 376th codon-edited cells showed higher PCNA expression and lower Bax-1 expression compared to controls.
Conclusions:
- Loss of Keap1 function confers significant antioxidative ability to Ad-MSCs.
- CRISPR/Cas9-mediated Keap1 gene editing is a promising strategy to enhance MSC viability for transplantation.
- This study highlights a novel application of CRISPR/Cas9 in improving stem cell therapeutics for conditions involving oxidative stress.
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