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Generation of TNFαR1 and ASIC1a Knockout Human Neural Stem Cells In Vitro by CRISPR/Cas9 System

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Abstract

Technologies based on autologous induced pluripotent stem cells (iPSCs) can become promising methods that provide tissue regeneration after a stroke which is currently one of the most acute social and medical problems. Transplantation of neural stem cells obtained from iPSCs by the method of directed differentiation can potentially stop the degradation of nerve tissue and significantly accelerate the regeneration processes. The advantage of using iPSCs is their ability to differentiate into various cell types. At the moment, there are proven methods for obtaining, cultivating and modifying these cells (genome editing), it is possible to create lines with various new properties. Knockout of genes for TNFaR1 and ASIC1a receptors can become such a genomic modification. TNFaR1 is the major receptor for Tumor Necrosis Factor (TNF), which is an essential multifunctional pro-inflammatory cytokine. TNFaR1 activation triggers the apoptosis program in the cell. ASIC1 is a transmembrane protein that is an essential component of acid-sensing ion channel (ASICs) complexes. These channels are receptor-activators of various intracellular systems (including apoptosis) in response to acidosis (lowered pH). Since strokes are accompanied by inflammatory and acidotic shock in the areas of nerve tissue damage, inactivation of the TNFaR1 and ASIC1 genes in the transplanted cells may increase cell survival and engraftment after transplantation. In this work, we describe the method of TNFaR1 and ASIC1a gene knockout using the CRISPR/Cas9 system in iPSCs, followed by the production of neural stem cells. The production of such knockout human neural stem cells (with the subsequent possibility of differentiation into neurons or glia) can also serve as the basis for fundamental studies of these genes, their products, and the basis for screening systems for drugs against stroke.

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ACKNOWLEDGMENTS

We thank the Core Centrum of Institute of Developmental Biology RAS for the opportunity to work on the equipment of the center.

Funding

The work was supported by the Ministry of Health of the Russian Federation within the framework of State Assignment nos. 121040600409-1 and 075-15-2019-1789 from the Ministry of Science and Higher Education of the Russian Federation, allocated to the Center for Precision Genome Editing and Genetic Technologies for Biomedicine. The part of work with cell staining and fluorescent microscopy was supported by the IDB RAS Government program of basic research no. 0108-2019-0004. The work of ASA was supported by Systems Biology Fellowship by Skoltech.

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Authors and Affiliations

  1. Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, ul. Oparina 4, 117198, Moscow, Russia

    A. S. Poltavets

  2. Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Ostrovitianov Street, 117997, Moscow, Russia

    N. V. Mescheryakova, A. S. Artyuhov & E. B. Dashinimaev

  3. Institute of Molecular Medicine, Sechenov First Moscow State Medical University, 119991, Moscow, Russia

    Yu. S. Kolesova

  4. Moscow Institute of Physics and Technology (State University), 141701, Dolgoprudny, Russia

    E. B. Dashinimaev

  5. Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 119334, Moscow, Russia

    E. B. Dashinimaev

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  1. A. S. Poltavets
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  2. N. V. Mescheryakova
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  3. Yu. S. Kolesova
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  4. A. S. Artyuhov
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  5. E. B. Dashinimaev
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Correspondence to A. S. Poltavets.

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Conflict of interest. The authors declared no conflict of interest.

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Poltavets, A.S., Mescheryakova, N.V., Kolesova, Y.S. et al. Generation of TNFαR1 and ASIC1a Knockout Human Neural Stem Cells In Vitro by CRISPR/Cas9 System. Neurochem. J. 15, 398–409 (2021). https://doi.org/10.1134/S1819712421040103

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  • DOI: https://doi.org/10.1134/S1819712421040103

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