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

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.