Generation of an induced pluripotent stem cell line, ICGi014-A, by reprogramming peripheral blood mononuclear cells from a patient with homozygous D90A mutation in SOD1 causing Amyotrophic lateral sclerosis

Ustyantseva, Elizaveta; Medvedev, S. P.; Ветчинова, А. С.; Иллариошкин, С.Н.; Leonov, Sergey; Zakian, Suren M.

doi:10.1016/j.scr.2019.101675

Cited by 6 publications

(8 citation statements)

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“…We utilized a previously described protocol of highly efficient motor neuron differentiation (Fig. 3a) [35] that allowed us to obtain iPSC-derived motor neurons with a characteristic morphology within 30 days. For each type of iPSC (K7-4Lf, iALS, SOD1-D91A, and SOD1-G128R), we differentiated three separate clones.…”

Section: Resultsmentioning

confidence: 99%

“…It is unclear how long MN should be cultured in the B- 27 deprived medium before the stress becomes decompensated and whether it will help to distinguish MN with different genotypes. Some authors use deficit media on astrocyte-neuron cultures, and probably this approach can be applied for MN monocultures as well [35, 64].…”

Section: Discussionmentioning

confidence: 99%

“…The iPSCs were seeded onto dishes coated with Matrigel-ESQ (Corning) in E8 (Gibco) medium and maintained in feeder-free conditions for at least 2 passages prior to differentiation. Motor neuron differentiation was performed according to the previously published 4-step protocol [35]. For neural patterning, the E8 medium was changed to basal neuronal differentiation medium (NDM): F12/DMEM:Neurobasal – 50:50, 0.5× N2 supplement, 0.5× B-27 supplement, 2 mM GlutaMAX, and 0.1 mM ascorbic acid.…”

Section: Methodsmentioning

confidence: 99%

“…The target mutation was further confirmed by Sanger sequencing (Supplementary Table 2). The clones used in the experiments were characterized according to the Human Pluripotent Stem Cell Registry standards with the protocols described earlier [34].…”

Section: Screening Of Ipsc Clones For the C272a>c (D91a) And C382g>c (G128r) Substitutionsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Screening Of Ipsc Clones For the C272a>c (D91a) And C382g>c (G128r) Substitutionsmentioning

confidence: 99%

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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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“…Indeed, this method of reprogramming blood cells into stem cells and expanding these cells for differentiation to various organs of interest may provide a useful tool to study patient-specific disease modeling and drug screening. Research groups have already shown examples generating somatic cells from PBMC-derived iPSCs that replicate genetic diseases in all kind of organs, for example, the brain (Frega et al, 2019 ; Ustyantseva et al, 2020 ), blood (Lyu et al, 2018 ), and heart (Perepelina et al, 2020 ). There are also promising examples of drug and toxicity screening (Chang et al, 2014 ; Elitt et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

Going Back and Forth: Episomal Vector Reprogramming of Peripheral Blood Mononuclear Cells to Induced Pluripotent Stem Cells and Subsequent Differentiation into Cardiomyocytes and Neuron-Astrocyte Co-cultures

Leeuw

Oostrom

Imholz

et al. 2020

Cellular Reprogramming

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Human induced pluripotent stem cells (iPSCs) can capture the diversity in the general human population as well as provide deeper insight in cellular mechanisms. This makes them suitable to study both fundamental and applied research subjects, such as disease modeling, gene-environment interactions, personalized medicine, and chemical toxicity. In an independent laboratory, we were able to generate iPSCs originating from human peripheral blood mononuclear cells according to a modified version of a temporal episomal vector (EV)-based induction method. The iPSCs could subsequently be differentiated into two different lineages: mesoderm-derived cardiomyocytes and ectoderm-derived neuron-astrocyte co-cultures. It was shown that the neuron-astrocyte culture developed a mature phenotype within the course of five weeks and depending on the medium composition, network formation and neuron-astrocyte cell ratios could be modified. Although previously it has been described that iPSCs generated with this EV-based induction protocol could differentiate to mesenchymal stem cells, hepatocytes, cardiomyocytes, and basic neuronal cultures, we now demonstrate differentiation into a culture containing both neurons and astrocytes.

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Induced pluripotent stem cells (iPSCs) as game‐changing tools in the treatment of neurodegenerative disease: Mirage or reality?

Yousefi

Abdollahii

Kouhbanani

et al. 2020

Journal Cellular Physiology

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Based on investigations, there exist tight correlations between neurodegenerative diseases' incidence and progression and aberrant protein aggregreferates in nervous tissue. However, the pathology of these diseases is not well known, leading to an inability to find an appropriate therapeutic approach to delay occurrence or slow many neurodegenerative diseases' development. The accessibility of induced pluripotent stem cells (iPSCs) in mimicking the phenotypes of various late‐onset neurodegenerative diseases presents a novel strategy for in vitro disease modeling. The iPSCs provide a valuable and well‐identified resource to clarify neurodegenerative disease mechanisms, as well as prepare a promising human stem cell platform for drug screening. Undoubtedly, neurodegenerative disease modeling using iPSCs has established innovative opportunities for both mechanistic types of research and recognition of novel disease treatments. Most important, the iPSCs have been considered as a novel autologous cell origin for cell‐based therapy of neurodegenerative diseases following differentiation to varied types of neural lineage cells (e.g. GABAergic neurons, dopamine neurons, cortical neurons, and motor neurons). In this review, we summarize iPSC‐based disease modeling in neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Moreover, we discuss the efficacy of cell‐replacement therapies for neurodegenerative disease.

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Generation of an induced pluripotent stem cell line, ICGi014-A, by reprogramming peripheral blood mononuclear cells from a patient with homozygous D90A mutation in SOD1 causing Amyotrophic lateral sclerosis

Cited by 6 publications

References 4 publications

A cell-based platform for oxidative stress monitoring in 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

Going Back and Forth: Episomal Vector Reprogramming of Peripheral Blood Mononuclear Cells to Induced Pluripotent Stem Cells and Subsequent Differentiation into Cardiomyocytes and Neuron-Astrocyte Co-cultures

Induced pluripotent stem cells (iPSCs) as game‐changing tools in the treatment of neurodegenerative disease: Mirage or reality?

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Generation of an induced pluripotent stem cell line, ICGi014-A, by reprogramming peripheral blood mononuclear cells from a patient with homozygous D90A mutation in SOD1 causing Amyotrophic lateral sclerosis

Cited by 6 publications

References 4 publications

A cell-based platform for oxidative stress monitoring in 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

Going Back and Forth: Episomal Vector Reprogramming of Peripheral Blood Mononuclear Cells to Induced Pluripotent Stem Cells and Subsequent Differentiation into Cardiomyocytes and Neuron-Astrocyte Co-cultures

Induced pluripotent stem cells (iPSCs) as game‐changing tools in the treatment of neurodegenerative disease: Mirage or reality?

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A cell-based platform for oxidative stress monitoring in 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