Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population

Malakhova, Anastasia A.; Григорьева, Э. В.; Павлова, С. В.; Malankhanova, T.B.; Валетдинова, К.Р.; Vyatkin, Yuri; Khabarova, Elena A.; Рзаев, Д. А.; Medvedev, S. P.

doi:10.1016/j.scr.2020.101952

Cited by 21 publications

(23 citation statements)

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Ustyantseva

Павлова

Malakhova

et al. 2022

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Ustyantseva

Павлова

Malakhova

et al. 2022

Sci Rep

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“…We introduced these mutations using CRISPR/Cas9, into a well-characterized control iPSC line (K7-4Lf), obtained earlier from a clinically healthy individual [30] (Supplementary Table 1) and recovered 66 clones for D91A variant and 124 clones for G128R variant. We screened those clones by tetra-primer ARMS PCR (for D91A) or qPCR with fluorescent probes (for G128R) and found 6 (9.1%) and 4 (3.2%) clones, respectively, presumably positive for the target mutations.…”

Section: Resultsmentioning

confidence: 99%

“…IPSC cells 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 [29] and a healthy individual (K7-4Lf) who had no associations with any genetic disease [30] (Supplementary Table 1) .…”

Section: Methodsmentioning

confidence: 99%

A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H₂O₂

Ustyantseva

Zakian

Medvedev

2021

Preprint

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Background: Oxidative stress plays an important role in the development of neurodegenerative diseases: it either can be the initiator or part of a pathological cascade leading to the neuron's death. Although a lot of methods are known for oxidative stress study, most of them operate on non-native cellular substrates or interfere with the cell functioning. Genetically encoded (GE) biosensors of oxidative stress demonstrated their general functionality and overall safety in various live systems. However, there is still insufficient data regarding their use for research of disease-related phenotypes in relevant model systems, such as human cells. Methods: We applied CRISPR/Cas9 genome editing to introduce mutations (c.272A>C and c.382G>C) in the associated with amyotrophic lateral sclerosis SOD1 gene of induced pluripotent stem cells (iPSC) obtained from a healthy individual. Using CRISPR/Cas9, we modified these mutant iPSC lines, as well as the parental iPSC line, and a patient-specific SOD1D91A/D91A iPSC line with ratiometric GE biosensors of cytoplasmic (Cyto-roGFP2-Orp1) and mitochondrial (Mito-roGFP2-Orp1) H2O2. The biosensors sequences along with a specific transactivator for doxycycline-controllable expression were inserted in the "safe harbor" AAVS1 (adeno-associated virus site 1) locus. We differentiated these transgenic iPSCs into motor neurons and investigated the functionality of the biosensors in such a system. We measured relative oxidation in the cultured motor neurons and its dependence on culture conditions, age, and genotype, as well as kinetics of H2O2 elimination in real-time. Results: We developed a cell-based platform consisting of isogenic iPSC lines with different genotypes associated with amyotrophic lateral sclerosis. The iPSC lines were modified with GE biosensors of cytoplasmic and mitochondrial H2O2. We provide proof-of-principle data showing that this approach may be suitable for monitoring oxidative stress in cell models of various neurodegenerative diseases as the biosensors reflect the redox state of neurons. Conclusion: We found that the GE biosensors inserted in the AAVS1 locus remain functional in motor neurons and reflect pathological features of mutant motor neurons, although the readout largely depends on the severity of the mutation.

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“…G-banding karyotyping was performed at passage 8 of EC and SMC as previously described ( Malakhova et al, 2020 ). We analyzed 50 metaphase spreads using Nikon microscope TiE with Lucia Cytogenetics software.…”

Section: Methodsmentioning

confidence: 99%

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches

Захарова

Saaya

Shevchenko

et al. 2022

Front. Bioeng. Biotechnol.

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In our previous study, we showed that discarded cardiac tissue from the right atrial appendage and right ventricular myocardium is an available source of functional endothelial and smooth muscle cells for regenerative medicine and tissue engineering. In the study, we aimed to find out what benefits are given by vascular cells from cardiac explants used for seeding on vascular patches engrafted to repair vascular defects in vivo. Additionally, to make the application of these cells safer in regenerative medicine we tested an in vitro approach that arrested mitotic division to avoid the potential tumorigenic effect of dividing cells. A tissue-engineered construction in the form of a patch based on a polycaprolactone-gelatin scaffold and seeded with endothelial and smooth muscle cells was implanted into the abdominal aorta of immunodeficient SCID mice. Aortic patency was assessed using ultrasound, MRI, immunohistochemical and histological staining. Endothelial and smooth muscle cells were treated with mitomycin C at a therapeutic concentration of 10 μg/ml for 2 h with subsequent analysis of cell proliferation and function. The absence of the tumorigenic effect of mitomycin C-treated cells, as well as their angiogenic potential, was examined by injecting them into immunodeficient mice. Cell-containing patches engrafted in the abdominal aorta of immunodeficient mice form the vessel wall loaded with the appropriate cells and extracellular matrix, and do not interfere with normal patency. Endothelial and smooth muscle cells treated with mitomycin C show no tumorigenic effect in the SCID immunodeficient mouse model. During in vitro experiments, we have shown that treatment with mitomycin C does not lead to a decrease in cell viability. Despite the absence of proliferation, mitomycin C-treated vascular cells retain specific cell markers, produce specific extracellular matrix, and demonstrate the ability to stimulate angiogenesis in vivo. We pioneered an approach to arresting cell division with mitomycin C in endothelial and smooth muscle cells from cardiac explant, which prevents the risk of malignancy from dividing cells in vascular surgery. We believe that this approach to the fabrication of tissue-engineered constructs based on mitotically inactivated cells from waste postoperative material may be valuable to bring closer the development of safe cell products for regenerative medicine.

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Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population

Cited by 21 publications

References 5 publications

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

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

A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H₂O₂

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches

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Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population

Cited by 21 publications

References 5 publications

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

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

A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H2O2

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches

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Product

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A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H₂O₂