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

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.


Results
Introduction of the SOD1 D91A and G128R mutations in iPSCs of the clinically healthy donor. The SOD1 gene has more than 140 mutations associated with ALS, which define the clinical features of the disease such as its manifestation age, rate of progression, presence of additional symptoms, etc. 30 . We chose c.272A>C and c.382G>C mutations that lead to either a relatively benign or severe disease course, respectively 31,32 . In the first step of our workflow (Fig. 1A), we designed corresponding CRISPR/Cas9 guide RNA targeting the sequences in the exons 4 and 5 of SOD1 and single-stranded oligodeoxynucleotides (ssODN) donor templates necessary for the introduction of c.272A>C and c.382G>C single-nucleotide mutations that lead to the D91A and G128R substitutions, respectively, in the SOD1 polypeptide (Fig. 1B, Supplementary Table S1).
We introduced these mutations, using CRISPR/Cas9, into a well-characterized control iPSC line (K7-4Lf), obtained earlier from a clinically healthy individual 33 (Supplementary Table S2) and recovered 66 clones for the D91A variant and 124 clones for the G128R variant of which 6 (9.1%) and 4 (3.2%) clones, respectively, were positive for the target mutations. As a result, several clones with different SOD1 allelic variants were obtained (Fig. 1C). Since we did not find any homozygous variants, we chose clones with SOD1 D91 A/del105 (iPSC line SOD1-D91A) and SOD1 G128R/K129* (iPSC line SOD1-G128R) variants for subsequent experiments (Supplementary Fig. S3). Importantly, these clones showed no mutations in five of the most likely predicted off-target sites ( Supplementary Fig. S4).

Generation of iPSC lines modified with genetically encoded biosensors of H 2 O 2 via CRISPR/
Cas9-mediated AAVS1 targeting. We introduced sequences of two genetically encoded biosensors, Cyto-roGFP2-Orp1, and Mito-roGFP2-Orp1, measuring H 2 O 2 levels in the cytoplasm and mitochondria respectively, in the genome of SOD1-D91A, SOD1-G128R, and the isogenic control line (K7-4Lf) to obtain stable expression. Additionally, we introduced the same sequences in the genome of a patient-specific iPSC line (iALS) previously generated in our lab from a person diagnosed with a hereditary form of ALS with a homozygous D91A mutation in SOD1 34 .
The Tet-On system, applied for the biosensors expression, consists of two elements: the biosensor's sequence under the control of the tetracycline-dependent promoter and the specific transactivator (rtTA, reverse tetracycline-controlled transactivator) essential for the controlled expression of the target genes 35 . To deliver these elements in the cell's genome, we used biallelic target insertion in the safe harbor AAVS1 locus via CRISPR/Cas9 ( Fig. 2A). The donor plasmids were either obtained from the vendor or constructed in our laboratory 36,37 (Supplementary Fig. S5). IPSC clones with the target insertions were selected using respective media and tested for the presence of the biosensors' expression in response to doxycycline (tetracycline derivative) addition (Fig. 2B). Selected clones were further examined for target and off-target insertions by PCR (Fig. 2C). Three clones for each cell line were selected for differentiation and analysis ( Supplementary Fig. S6).
Neuronal differentiation of the modified iPSC lines and the biosensor expression in iPSC-derived motor neurons. We utilized a previously described protocol of highly efficient MN differentiation 38 . All iPSC-derived MNs stained positively for the most common markers: choline acetyltransferase (ChAT), ISL LIM homeobox 1 (ISL1), and motor neuron and pancreas homeobox (MNX1), and expressed mRNA of these proteins ( Supplementary Fig. S7). Differentiation efficiency, analyzed on day 20 of differentiation by counting the ISL-positive cells using flow cytometry, showed 89-95% of MNs in the samples (Fig. 3A).
To characterize MNs obtained, we measured the axonal length of the iPSC-derived MNs on day 21 of differentiation. SOD1-D91A (90.7 ± 42.6 μm) and iALS (90.4 ± 41.9 μm) MNs had considerably shorter axonal processes compared to the control K7-4Lf MNs (107.8 ± 45.5 μm). The mean axon length in SOD1-G128R (67 ± 30.6 μm) MNs was even lower, suggesting a highly disturbed function of these cells (Fig. 3B). The AAVS1 site is located in the intron of a transcriptionally active gene and was described previously as suitable for stable expression of transgenes 39 . However, we have discovered that the MNs did not always retain a detectable fluorescence level of the biosensor at the terminal stages of differentiation, and this did not depend on a particular cell line (Fig. 3C,D). Analysis of the expression level of rtTA and roGFP2 in the MNs with low intensity of the biosensor signal revealed that the terminally differentiated MNs expressed mRNA of the rtTA at the same level as the corresponding iPSCs from which they were obtained. At the same time, the expression of the biosensor roGFP2 was decreased by two orders, suggesting that the promoter of the biosensor was selectively inhibited (Fig. 3E). We performed differentiation, supplementing the medium regularly with doxycycline from the first day of differentiation to keep the biosensor promoter in an active state. As a result, the biosensor retained high signal intensity in the differentiated MNs as well as mRNA expression on a level comparable to the iPSCs (Fig. 3E). Moreover, the dynamic range (difference between fully oxidized and fully reduced biosensor's state) of the signals generated in the MNs was relatively stable across the lines, suggesting a similar level of the biosensor expression ( Supplementary Fig S8). Nutrient deprivation affects the mitochondrial level of H 2 O 2 regardless of the genotype. The basal level of H 2 O 2 reflects stationary redox balance and the general condition of the cell. The H 2 O 2 biosensor allows us to estimate the relative amount of H 2 O 2 molecules in the compartment and determine if it is different from the control. We performed live imaging of the mature MNs (Day 29) at the end of the differentiation protocol to obtain information about their redox state. We did not observe any signs of pathological oxidation in the cytoplasm and mitochondria of the SOD1-D91A and iALS MNs. SOD1-G128R MNs, however, demonstrated Induced pluripotent stem cell (iPSC) lines with SOD1 mutations were generated either from iPSCs of a healthy donor by CRISPR/Cas9-mediated genome editing or obtained from a patient with ALS. The iPSCs from a healthy donor served as an isogenic negative control (Neg. Ctrl); patient-specific iPSCs served as a positive control (Pos. Ctrl). Next, the iPSC lines (control, SOD1-mutated, and patient-specific) were modified with biosensors of cytoplasmic (Cyto-roGFP2-Orp1) and mitochondrial (Mito-roGFP2-Orp1) H 2 O 2 by targeted insertion of the biosensors' sequences with CRISPR/Cas9. Then, the modified iPSC lines were differentiated in spinal motor neurons with subsequent analysis of the redox state of the neurons with the biosensors. (B) Schematic of the SOD1 gene with partial sequences of exons 4 and 5. Protospacers designed for CRISPR/Cas9mediated double-stranded breaks are underlined with black lines, protospacer adjacent motif (PAM)-with green lines; target mutations are in bold and marked with black triangles. (C) Partial SOD1 sequences of exons 4 and 5 of the SOD1-D91A and SOD1-G128R iPSC lines. Int. 3-intron 3, substituted nucleotides marked with an arrow, corresponding mutated amino acids are highlighted in red. The box contains a list of clones with alternative SOD1 variants obtained in the study.  (Fig. 4A). Notably, the cytoplasmic oxidation measured at the stage of immature MNs (Day 20) was similar for all neurons, suggesting that the SOD1-G128R MNs hyper oxidation develops with the maturation of the MNs (Fig. 4B). To correct the observed phenotype, we added a combination of neurotrophic factors (NTFs, See Materials and Methods) to the culture medium during SOD1-G128R MNs maturation (differentiation days [19][20][21][22][23][24][25][26][27][28][29]. This resulted in a significant decrease in cytoplasmic H 2 O 2 to the normal level. However, it did not affect the mitochondrial level of H 2 O 2 (Fig. 4C).
To put additional stress on the MNs, we depleted culturing medium from the majority of the nutrients by removing the B-27 supplement (chemically-defined mixture of antioxidant enzymes, proteins, vitamins, and fatty acids) 24 h before live imaging and measured the cytoplasmic and mitochondrial H 2 O 2 levels. We discovered that B-27 deprivation did not influence the cytoplasmic level of H 2 O 2 (Fig. 4A,D, Supplementary Fig. S9). However, MNs cultured in the absence of B-27 demonstrated a significant increase in the mitochondrial H 2 O 2 level, which was, on average, four times higher than in the cytoplasm of the corresponding neurons. For the SOD1-G128R MNs, the B-27 deprivation resulted in an additional increase in both cytoplasmic and mitochondrial H 2 O 2 levels (Fig. 4D, Supplementary Fig. S9).
Interestingly, the H 2 O 2 levels in the cytoplasm and mitochondria of mature iALS MNs were lower compared to the control, probably due to the different origin of the iALS cell line (Fig. 4A,D).
Antioxidants removal from the medium induces H 2 O 2 accumulation in the cytoplasm of motor neurons. Amongst many additives, the neuronal medium contains antioxidants, such as ascorbic acid, vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione, acting against ROS that appear in the medium during in vitro culturing. The removal of the antioxidants from the medium may force MNs to rely on endogenous antioxidant systems for ROS neutralization, making the mutant MNs more vulnerable. We cultured the neurons in the antioxidant-free medium (without ascorbic acid, and using the B-27 supplement without antioxidants) for three days in addition to the differentiation protocol and performed live imaging of the MNs (Day 32, Fig. 5A,B). All MNs showed an increase in the cytoplasmic H 2 O 2 level by 1.5-2.5 times. However, only in the SOD1-G128R MNs the mitochondrial level of H 2 O 2 increased along with the cytoplasmic level. In addition, SOD1-G128R MNs also demonstrated visible changes in the morphology with the axon attrition and cytoplasmic vacuolization (Fig. 5C). Culturing of SOD1-G128R MNs with the NTFs reduced oxidation of the

Cyto-roGFP2-Orp1 biosensor, expressed in motor neurons, reflects the kinetics of H 2 O 2 neutralization.
To assess the dynamic response of the MNs expressing Cyto-roGFP2-Orp1 and Mito-roGFP2-Orp1 biosensors to oxidation, we first determined the concentration of H 2 O 2 that did not affect MN viability in the culture. Only treatment with 10 µM H 2 O 2 did not significantly affect the viability of the tested MNs (Supplementary Fig. S10), which was consistent with the previously published data 40,41 . Next, we tested whether culturing in the standard neuronal medium distorts the cellular response to the H 2 O 2 and performed a live recording of the Cyto-roGFP2-Orp1-expressing MNs reaction to the addition of 10 μM H 2 O 2 . These MNs were either cultured in the standard differentiation medium or starved in the nutrient-deprived medium before the measurement (Fig. 6A). The overall reaction of the cells was similar: MNs expressing Cyto-roGFP2-Orp1 displayed oxidation followed by slow reduction, reflecting the change in the cytoplasmic H 2 O 2 level. However, the reaction of MNs cultured in the nutrient-deprived medium before the experiment was more prominent. We detected a higher delta of the biosensor oxidation and faster reduction compared to the non-starved MNs (Fig. 6B,C). Since components present in the standard medium affected the cellular reaction, we conducted further measurements of the dynamic response on the cells that were starved before the experiment. Using the parameters established earlier, we recorded the reaction of MNs expressing the Mito-roGFP2-Orp1 biosensor to 10 μM H 2 O 2 in realtime. We did not detect any response of the Mito-roGFP2-Orp1 biosensor to the exogenous H 2 O 2 . An oxidation value of the biosensor remained constant during imaging, suggesting that the mitochondrial H 2 O 2 level was also stable (Fig. 6D). The addition of H 2 O 2 in higher concentrations (25 μM and 50 μM) induced mitochondrial oxidation but damaged the neurons. With this finding, we decided not to measure the dynamic response for the Mito-roGFP2-Orp1 sensor (Fig. 6E). Next, we analyzed how the mutations introduced in SOD1 affected neuronal reaction to exogenous H 2 O 2 . We did not find any differences in H 2 O 2 utilization in the cytoplasm of Cyto-roGFP2-Orp1-expressing SOD1-D91A, iALS, and control (K7-4Lf) MNs ( Fig. 6F-H). Analysis of the SOD1-G128R MNs response to the H 2 O 2 revealed an aberrant reaction. Due to the high initial oxidation of the MNs, cells did not respond to the exogenous H 2 O 2 ( Fig. 6F-H). Although culturing of the SOD1-G128R MNs with the NTFs slightly reduced initial oxidation of the cytoplasm, it did not affect the cellular reaction to the H 2 O 2. The Cyto-roGFP2-Orp1-expressing MNs demonstrated moderate oxidation of the cytoplasm without signs of subsequent reduction (Fig. 6I).
Motor neurons expressing the Cyto-roGFP2-Orp1 biosensor accumulate H 2 O 2 in the cytoplasm due to glutamate-induced excitotoxicity. Glutamate excitotoxicity is a known pathological hallmark   42 . To test whether the Cyto-roGFP2-Orp1 and Mito-roGFP2-Orp1 biosensors can reflect redox imbalance caused by the excitotoxicity, we incubated MNs, expressing these biosensors with monosodium glutamate (20 μM) and the glutamate reuptake inhibitor (PDC, 100 μM) for five days and measured the cytoplasmic and mitochondrial H 2 O 2 levels. Since SOD1-G128R MNs died shortly after the beginning of the experiment due to reduced viability, the measurement was conducted only for K7-4Lf, SOD1-D91A, and iALS MNs. We discovered that the glutamate treatment induced the accumulation of H 2 O 2 in the cytoplasm regardless of the MN genotype (Fig. 7A). Cytoplasmic oxidation in iALS MNs treated with glutamate was higher compared to the glutamate-treated control MNs. However, we did not find the same for the SOD1-D91A MNs (Fig. 7A), indicating that this effect was not due to the SOD1 mutation. We did not observe the same for the mitochondria, despite the known connection of mitochondrial dysfunction with excitotoxicity ( Fig. 7A) 43 . The mitochondrial oxidation in SOD1-D91A MNs in both control and glutamate treated samples was increased (Fig. 7B), although we were unable to determine if the oxidation was a hallmark of the SOD1 mutation or a technical artifact. Further, we investigated how incubation with monosodium glutamate affected the dynamics of H 2 O 2 utilization in the cytoplasm of the Cyto-roGFP2-Orp1-expressing MNs. We found that MNs treated with glutamate had a reduced recovery rate after the H 2 O 2 addition compared to the non-treated sample (Fig. 7C-G).

Discussion
Nowadays, genetically encoded biosensors are frequently applied to research physiological and pathological processes 44,45 , replacing molecular probes. Indeed, they have provided extensive information about the dynamics of redox balance components, e.g., H 2 O 2 or GSH/GSSG ratio, in different cell compartments and tissues 22,46 .  (Fig. 1A). This approach allowed us to obtain iPSC lines, stably expressing the biosensors, and avoid potential undesirable effects of random integration (Fig. 2). For the first time, to our knowledge, we showed that the biosensor expressing in such a system is a reliable method to study MNs in vitro, as the intensity of the signal generated from a single copy of the biosensor and the dynamic range of that signal, are sufficient for routine imaging (Supplementary Fig. S8). However, we found that the AAVS1 locus does not support the stable expression of the integrated biosensors in MNs despite its "safe harbor" status, probably, due to chromatin remodeling occurring during differentiation 47 . Although constant activation of the biosensor promoter during differentiation, applied here, prevented it from silencing, this in itself emphasizes the necessity to discover new safe harbors in the human genome ( Fig. 3C-E). The measurements, conducted in MNs with Cyto-roGFP2-Orp1 and Mito-roGFP2-Orp1 biosensors, revealed that the basal level of cytoplasmic and mitochondrial oxidation is dynamic and serves as an indicator of cellular distress. Nutrient deprivation, known to induce moderate stress, activates autophagy through the ROS signaling [48][49][50] . In our data, MNs deprived of nutrients demonstrated additional oxidation of the mitochondria (Fig. 4), which was in line with the previously published data 48 . Additionally, a stable cytoplasmic level of H 2 O 2 in this condition also suggests that this is a part of a natural response. Interestingly, despite the stability of the cytoplasmic H 2 O 2 level in the nutrient-deprived MNs, the reaction of the cells to exogenous H 2 O 2 was more prominent. Specifically, a higher amplitude of oxidation and faster recovery were recorded (Fig. 6A-C), implying www.nature.com/scientificreports/ that in these moderate stress conditions, the buffering capacity of the cytoplasm is lower, but the antioxidant system is in the mobilized, more active state 51 . Differences in cytoplasmic and mitochondrial reactions to the exogenous H 2 O 2 reflect relative independence of the mitochondrial and cytoplasmic antioxidant systems (Fig. 6D-E). The absence of significant changes in the signal of the Mito-roGFP2-Orp1 sensor and the presence of a normal reaction of the Cyto-roGFP2-Orp1 sensor in response to 10 μM H 2 O 2 suggests that the cytoplasmic antioxidant system neutralizes most exogenous H 2 O 2 molecules. The reaction that only appears in response to a lethal amount of H 2 O 2 suggests that exceeding hydrogen peroxide level in the cytoplasm above the value that it can quickly neutralize leads to a transfer of H 2 O 2 molecules into mitochondria and apoptosis induction 52 .  (A, B, F, G) To test if the developed platform can reflect pathological oxidation in MNs caused by a genetic mutation, we generated isogenic cell lines with SOD1 mutations of, presumably, different severity: D91A and G128R 31,32 . Since no homozygous variants have been found, we selected the lines that have one allele with the target mutation and the other with large deletion (SOD1-D91A) or premature termination codon (SOD1-G128R) to maximize potential damage and make the pathological phenotype more perceptible (Fig. 1B,C). While the neurons generated from iPSC lines with a D91A mutation were not different from the control, MNs derived from the SOD1-G128R iPSCs demonstrated higher levels of oxidation in both the cytoplasm and mitochondria, which only increased in stress conditions, such as nutrient or antioxidant deprivation (Figs. 4, 5, 6). Through live imaging of the neurons in the different stages of differentiation, we were able to detect that described pathological features only appear in the latest stages of differentiation (Fig. 4B), which is consistent with the fact that ALS does not manifest until a certain age 53 . Using the separate measurement of H 2 O 2 in different compartments, we were able to detect that mitochondrial oxidation in mature SOD1-G128R MNs was more pronounced than that of the cytoplasm (Figs. 4A, 5A,B), suggesting that accumulation of H 2 O 2 in mitochondria precedes its accumulation in the cytoplasm. Together with the slower axon growth (Fig. 3B), this could be a sign of mitochondrial dysfunction that has been linked to neurodegeneration before 1,54 . Moreover, the inability of neurotrophic factors to correct this phenotype indicates the severity and irreversibility of the cellular condition observed at this point (Figs. 4C, 5D).
Glutamate excitotoxicity is one of the major mechanisms of ALS development, in which excessive activation of the glutamate receptors leads to mitochondrial dysfunction and apoptosis 42,52 . It is known that this process is accompanied by increased ROS production, which connects it with oxidative stress 55 . In our study, we, for the first time, observed in live MNs how glutamate-induced excitotoxicity affected oxidation in the neuronal compartments. Glutamate-treated neurons demonstrated accumulation of H 2 O 2 in the cytoplasm and a slower recovery rate after H 2 O 2 addition (Fig. 7). However, no differences between the D91A mutant and control MNs were observed, suggesting that the power of stress applied or the degree of MN maturation was insufficient for the pathological phenotype to manifest, meaning that further optimization of the study parameters is required.
In conclusion, our work presents a new approach for the application of cell-based disease models for research that can be used to generate other similar models. We expect this approach to be expanded to include other disease-associated mutations or the biosensors of other cellular processes. There are, however, still elements that require further optimization, such as research for the new safe-harbor loci that sustain transgene expression. Moreover, the culture conditions applied for the study must be considered since modern cell culture systems are highly protective and can hinder the results of the measurements. We also look forward to the application of automated cell imaging, allowing continuous measurement during cell differentiation and maturation, which could also be beneficial for more complex research and screening for future therapeutics.

Methods
IPS cell culture. iPSCs were maintained on the layer of mitotically inactivated mouse embryonic fibroblasts in KnockOut DMEM (Gibco) with 15% knockout serum replacement (Gibco), 0.1 mM non-essential amino acids (Gibco), penicillin/streptomycin (Lonza), 1 mM GlutaMAX-I, and 10 ng/mL bFGF at 37 °C and 5% CO 2 . IPSCs were dissociated with TrypLE (Gibco) and split at 1:10 twice a week in the iPSC medium supplemented with 10 ng/ml Y-27632. Original iPSC lines were derived from the patient (iALS) with a diagnosed hereditary form of ALS 37 and a healthy individual (K7-4Lf) who had no associations with any genetic disease 33 (Supplementary Table S2). The use of the iPSC lines, generated from the patients' materials, in the study has been approved by the Research Ethics Committee of FSBI Federal Neurosurgical Center (Novosibirsk, Russia), protocol number 1 14/03/2017.

SOD1.
The guide RNAs (gRNAs) targeting sequences in exons 4 and 5 of the SOD1 gene and the AAVS1 locus were designed using the web-based tool http:// crispr. mit. edu 56 . The Alt-R ® crisprRNA and tracrRNA were obtained from IDT (Integrated DNA technologies), and Cas9 protein was expressed in E. coli and purified according to the previously published protocol 57 . The CRISPR/Cas9 ribonucleoprotein (RNP) complexes were assembled according to the manufacturer's instructions on the day of transfection. For the introduction of the c.272A>C and c.382G>C mutations we used appropriate RNP (20 pmol tracrRNA + 20 pmol crisprRNA (SOD1-4/SOD1-5) + 20 pmol Cas9) complexes mixed with 100 pmol of D91A ssODN (single-stranded oligodeoxynucleotide) or G128R ssODN donor. The cells were passed 24 h before the transfection in the iPSC medium supplemented with Y-27632 (10 ng/ml). Transfection was performed using Neon Transfection System 10 μl Kit (Invitrogen) according to the manufacturer's instructions. The cells were seeded on feeder-coated 4 cm 2 dishes in the iPSC medium supplemented with Y-27632 (10 ng/ml). The next day, the cells were dissociated with TrypLE, strained through the cell strainer, and subcloned on 96-well plates for propagation and analysis. Genomic DNA of the survived clones was obtained and analyzed for the presence of the target mutations.
To detect the c.272A>C mutation, we designed primers for tetra-primer ARMS (amplification-refractory mutation system) PCR screening using http:// prime r1. soton. ac. uk/ prime r1. html 58 and performed touchdown 3-step PCR with annealing at 68-64 °C for 9 cycles, then at 64 °C for 21 cycles. The PCR products were analyzed in 2% agarose gel. Clones positive for the mutant allele were further examined by Sanger sequencing. To detect the c.382G>C mutation, we designed a pair of primers that amplify the target locus of the SOD1 gene and two fluorescent probes targeting either wild-type or mutant sequences. Using LightCycler 480 (Roche), we analyzed the clones and selected those who had strong signals from the mutant-targeted probe. The target mutation was further confirmed by Sanger sequencing. Potential off-target CRISPR/Cas9 sites were determined using the Benchling algorithm (https:// www. bench ling. com/), and the top 5 hits were then investigated in the obtained cell lines by Sanger sequencing. Clones used in the experiments were characterized according to the Human

Generation of iPSC lines with target AAVS1 inserts.
To insert Cyto-roGFP2-Orp1, Mito-roGFP2-Orp1 and transactivator in AAVS1, we used AAVS1 RNP (100 pmol tracrRNA + 100 pmol AAVS1 crRNA + 100 pmol Cas9) mixed with 5 μg of donor plasmids mix, containing equimolar amounts of transactivator donor (pAAVS1-Neo-M2rtTA, Addgene # 60843) + pCyto-roGFP2-Orp1-donor or pMito-roGFP2-Orp1donor. The transfection was performed using Neon Transfection System 100 μl Kit according to instructions. The cells were then seeded on feeder-coated 10 cm 2 dishes in the iPSC medium supplemented with Y-27632 (10 ng/ml) and maintained before the selection until small colonies formed. We supplemented the iPSC medium with puromycin dihydrochloride for 3 days to select subclones with the target inserts. Then we replaced the antibiotic with neomycin sulfate and incubated the cells for 4-5 more days. Antibiotic concentrations were determined for each cell line before the experiment. At the end of the selection, we added doxycycline hyclate (2 μg/ ml) and examined the remained clones for the presence of fluorescent signal from the biosensors' roGFP2 using the Nikon Eclipse Ti2-E microscope. The clones positive for roGFP2 expression that survived double antibiotic selection were manually harvested into separate dishes for maintenance and analysis. Genomic DNA extracted from these iPSC clones was analyzed for the presence of the target and off-target inserts of the donor plasmids using PCR (Supplementary Table S1).
Co-localization of the roGFP2 fluorescent signal with the cytoplasm and mitochondria. Immunocytochemistry. The cells were fixed in 4% formaldehyde solution for 10 min at room temperature (RT), permeabilized with 0.5% Triton X-100 for 30 min at RT, and then incubated with blocking buffer (1% bovine serum albumin (BSA) in PBS) for 30 min at RT. After, the cells were incubated with specific primary antibodies overnight at 4 °C. Next, the appropriate secondary antibodies were added for 1.5-2 h incubation at RT. All antibodies were diluted in blocking buffer, and the cell nuclei were visualized with DAPI (1 μg/ml solution in PBS). The antibodies and their dilution ratios are listed in Supplementary Table S11. Micrographs were captured using either Nikon eclipse Ti-E microscope (Nikon) and NIS Elements software or the LSM-780 (Zeiss) microscope and ZEN black software.

Reverse-transcription quantitative PCR (RT-qPCR
Flow cytometry analysis. The cells were dissociated with Accutase on day 20 of the differentiation protocol, resuspended in cold PBS, and centrifuged at 400g for 5 min (the same settings were used for all subsequent centrifugation steps). The pellet was resuspended in 1 ml cold 4% formaldehyde solution and incubated on ice for 10-15 min. Next, we added 1 ml cold PBS, centrifuged the cells, discarded supernatant, resuspended the pellet in 1 ml ice-cold 100% methanol, and incubated it for 10-15 min on ice. Then, the pellet was washed twice with flow cytometry staining buffer (1% BSA, 0.2 μM EDTA, in PBS) and resuspended in it to 1 × 10 6 cells/ml concentration. 100 μl of the cell suspension (1 × 10 6 cells/ml) was incubated with anti-ISL primary antibodies overnight at 4 °C. The cells were washed with flow cytometry staining buffer, incubated with the secondary antibodies for 30 min at RT, and analyzed using FACSAria (BD Biosciences). Unlabeled cells and isotype-labeled cells were used as controls.
Fluorescence intensity measurement. MN images (four for each sample) were obtained using the Zeiss LSM-780 confocal laser scanning microscope (Pan-Apochromat 20 × objective) adjusted for visualization of a green dye (excitation-488 nm; emission collection at 500-530 nm). Mean intensity of the fluorescence on each image was measured using the ImageJ software and corrected total cell fluorescence (CTCF) was calculated with the formula: CTCF = Integrated Density − (Area occupied by cells × Mean fluorescence of background readings).
Axon measurement. Immature MNs were seeded on the cell imaging coverglasses and grown for two days in a medium supplemented with a neurotrophic factor (NTF) cocktail: IGF1 (PeproTech, 10 ng/ml), CNTF (PeproTech, 10 ng/ml), and BDNF (PeproTech, 10 ng/ml). Doxycycline (2 μg/ml) was added to induce the roGFP2 expression. Images were obtained with the Nikon Eclipse Ti-2E microscope (20 × objective, FITC channel). Using ImageJ, we manually measured the length of the longest processes (axons) of free-lying neurons with visible ends. Only axons with a length more than twice the size of the neurons' bodies were considered for the analysis. If the neuron had two long processes, the longest one was considered for the measurement. The mean length of the axons was calculated based on the data obtained from the differentiation of three separate iPSC clones for each genotype.
Image acquisition. The general procedures for the image acquisition and data analysis, including samples and solution preparations, microscopy settings, biosensors' calibration, basal H 2 O 2 measurement, H 2 O 2 utilization analysis, and data normalization, are described in the "Supplementary methods".
XTT viability assay. The viability of the MNs was assessed 3 h after the addition of H 2 O 2 in different concentrations (10 μM, 25 μM, 50 μM, and 100 μM) by an XTT test with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (Roche), according to the manufacturer's instructions at 20 h after the reagents were added. Cells were seeded on Matrigel-covered 96-well plates (1.6 × 10 4 cells/well). Before adding H 2 O 2 , we replaced the neuronal medium with HBSS + Ca 2+ + Mg 2+ . For each H 2 O 2 concentration, the experiments were carried out in three replicates. The viability data were normalized to the values obtained in the control well, treated with PBS and analyzed by Welch t-test.
Excitotoxicity induction assay. MNs were incubated in the neuronal maintenance medium supplemented with 20 μM monosodium glutamate (Sigma-Aldrich) and 100 μM l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, Sigma-Aldrich) for 5 days, changing the medium every other day. After 5 days of incubation, we obtained images of the treated MNs and non-treated control MNs. The data obtained at the end of the experiment were normalized to the starting oxidation values, measured before the glutamate addition, to describe the changes that emerged during the experiment.
Statistics. Graphs and statistical analyses were performed in GraphPad Prism, version 9.2.0 (https:// www. graph pad. com/ scien tifc-sofwa re/ prism/). Statistical analyses were performed using Welch t-test for single-pair comparisons or one-way ANOVA with post hoc Tukey's or Dunnett's tests for multiple comparisons, where applicable.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.