Oxidative stress monitoring in iPSC-derived motor neurons using genetically encoded biosensors of H2O2

Abstract

Oxidative stress plays an important role in the development of neurodegenerative diseases, being either the initiator or part of a pathological cascade that leads to the neuron’s death. Genetically encoded biosensors of oxidative stress demonstrated their general functionality and overall safety in various systems. However, there is still insufficient data regarding their use in the research of disease-related phenotypes in relevant model systems, such as human cells. Here, we establish an approach for monitoring the redox state of live motor neurons with SOD1 mutations associated with amyotrophic lateral sclerosis. Using CRISPR/Cas9, we insert genetically encoded biosensors of cytoplasmic and mitochondrial H2O2 in the genome of induced pluripotent stem cell (iPSC) lines. We demonstrate that the biosensors remain functional in motor neurons derived from these iPSCs and reflect the differences in the stationary redox state of the neurons with different genotypes. Moreover, we show that the biosensors respond to alterations in motor neuron oxidation caused by either environmental changes or cellular stress. Thus, the obtained platform is suitable for cell-based research of neurodegenerative mechanisms.

A Model of iPSC-Derived Macrophages with TNFAIP3 Overexpression Reveals the Peculiarities of TNFAIP3 Protein Expression and Function in Human Macrophages

“…Specifically, Zhu and co-authors discovered that during the development of transgenic mice with rtTA expression, a stably integrated Tet-On promoter was silenced in mouse neurons, probably due to a decrease in chromatin accessibility for a tetracycline transactivator [ 79 ]. Ustyantseva and co-authors [ 80 ] showed that human motor neurons differentiated in vitro from iPSCs with a DOX-inducible expression of H 2 O 2 biosensors lost biosensor expression, but a regular supplementation of the differentiation medium with DOX helped to overcome the problem. Our data extend these observations to iPSC-derived macrophages.…”

“…The A20 encoding nucleotide sequence was obtained by PCR from pEGFP-C1-A20 (Addgene #22141) using a Q5 ® High-Fidelity DNA Polymerase and primers ( Supplementary Table S2 ). Plasmid assembly was performed using standard molecular cloning methods by replacing the MluI-AsiGI fragment in the pCyto-roGFP2-Orp1-donor plasmid [ 80 ] with the A20 PCR product. The pAAVS1-TRE-CMV-TNFAIP3 construct assembly was confirmed by restriction analysis and Sanger sequencing (SB RAS Genomics Core Facility, Novosibirsk, Russia).…”

“…On the next day, the cells were dissociated using TrypLE, counted using a Countess™ automated cell counter (Thermo Fisher Scientific, CA, USA), and 10 6 cells were transfected with an equimolar mix of the three donor plasmids (5 µg total) using the Neon™ Transfection System 100 μL Kit (Thermo Fisher Scientific) (one pulse, 30 ms, 1100 V). The following plasmids were used for K7-iPSC electroporation: (i) pX458-AAVS1 encoding guide RNA for human AAVS1 locus and SpCas9 protein [ 80 ]; (ii) AAVS1-Neo-M2rtTA (Addgene #60843) containing a constitutive M2rtTA expression cassette and neomycin resistance gene; and (iii) the generated pAAVS1-CMV-TRE-hTNFAIP3 plasmid containing A20 gene cDNA under a tetracycline-inducible promoter ( Supplementary Table S1 ; Figure 1 ).…”

Detection of ER Stress in iPSC-Derived Neurons Carrying the p.N370S Mutation in the GBA1 Gene

“…After selection, surviving colonies were mechanically transferred to 48-well plates. Transgene integration was analyzed by PCR using primers (Table 2) as previously described [35].…”

Effect of M694V mutation in the MEFV gene, associated with Familial Mediterranean fever, on the morphology of iPSC-derived macrophages

“…The main advantage of iPSCs is the almost unlimited ability of cultivation and differentiation serving as a proper source of pluripotent stem cells and any type of cells in the living organism. IPSCs were successfully used for studying autoinflammatory [17,18], neurodegenerative [17][18][19][20][21][22][23][24], and other diseases. There are few attempts to create iPSCs for FMF patients, for example, a Turkish patient with a homozygous missense mutation (p.Met694Val) in the MEFV gene [25].…”