Generation of iPSC lines from two patients affected by febrile seizure due to inherited missense mutation in SCN1A gene

Scalise, Stefania; Scaramuzzino, Luana; Lucchino, Valeria; Esposito, Claudia; Malatesta, Paola; Grillone, Katia; Perrotti, Nicola; Cuda, Giovanni; Parrotta, Elvira Immacolata

doi:10.1016/j.scr.2020.102083

Cited by 14 publications

(10 citation statements)

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“…We are presently using the integrated approach described above to analyze hiPSC-NSCs from a family with an inherited form of febrile seizures secondary to a specific SCN1A gene mutation. [64] We aim to dissect the molecular mechanisms underlying the disease and to bring novel information on the structural changes produced by this mutation on the morphology of the nuclear channel. [64,65] Besides other important applications, this method can be extended to other channelopathies involving nervous, cardiovascular, respiratory, urinary systems that may arise as a consequence of ion channel defects.…”

Section: Discussionmentioning

confidence: 99%

“…[64] We aim to dissect the molecular mechanisms underlying the disease and to bring novel information on the structural changes produced by this mutation on the morphology of the nuclear channel. [64,65] Besides other important applications, this method can be extended to other channelopathies involving nervous, cardiovascular, respiratory, urinary systems that may arise as a consequence of ion channel defects. Together with this multiple characterization approach we also present a new theoretical-computational method with the aim of identifying the Na v channel among the many particles found in the TEM imaging frames.…”

Section: Discussionmentioning

confidence: 99%

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Direct Visualization and Identification of Membrane Voltage‐Gated Sodium Channels from Human iPSC‐Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy

et al. 2022

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In this study, transmission electron microscopy atomic force microscopy, and surface enhanced Raman spectroscopy are combined through a direct imaging approach, to gather structural and chemical information of complex molecular systems such as ion channels in their original plasma membrane. Customized microfabricated sample holder allows to characterize Nav channels embedded in the original plasma membrane extracted from neuronal cells that are derived from healthy human induced pluripotent stem cells. The identification of the channels is accomplished by using two different approaches, one of them widely used in cryo‐EM (the particle analysis method) and the other based on a novel Zernike Polynomial expansion of the images bitmap. This approach allows to carry out a whole series of investigations, one complementary to the other, on the same sample, preserving its state as close as possible to the original membrane configuration.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Direct Visualization and Identification of Membrane Voltage‐Gated Sodium Channels from Human iPSC‐Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy

et al. 2022

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“…In addition to these properties, PSCs also have the ability to give rise to almost any cell type, both in vivo and in vitro. On the basis of these key features, PSCs technology holds enormous potential in many fields of biomedical research, including drug screening, development and repurposing [62][63][64][65], disease modelling [66][67][68][69][70], cell therapy [71], aging [72], and regenerative medicine [73]. Although significant progress has been made, many hurdles still need to be overcome to push PSCs technology towards the road to clinical practice.…”

Section: Discussionmentioning

confidence: 99%

Uncovering the Metabolic and Stress Responses of Human Embryonic Stem Cells to FTH1 Gene Silencing

Scaramuzzino

Lucchino

Scalise

et al. 2021

Cells

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Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs’ response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.

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“…In addition to these properties, PSCs also have the ability to give rise to almost any cell type, both in vivo and in vitro. On the bases of these key features, the PSCs technology holds enormous potential to attempt many research and biomedical applications, including drug screening, development, and repurposing (5,16,78,89), disease modelling (47,(54)(55)(56)70), cell therapy (82), aging (59), and regenerative medicine (57). Although signi cant progress has been made, many hurdles still need to be overcome to push PSCs technology towards the road to clinical practice.…”

Section: Discussionmentioning

confidence: 99%

Dissecting the Molecular Response of Human Escs to Iron-Mediated Oxidative Stress by Genetic Silencing of FTH1 Gene

Scaramuzzino¹,

Lucchino²,

Scalise³

et al. 2021

Preprint

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Background: Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. As such, to function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated and characterized different aspects involved in ESCs response to iron accumulation following stable knockdown of ferritin heavy chain (FTH1) gene, encoding for a major iron storage protein with ferroxidase activity. Methods: stable FTH1 knockdown of H9-hES cell line was achieved with the use of shRNA lentiviral particles. Upon FTH1 silencing, we speculated whether hESCs still retained their pluripotency capability were first monitored for their capability the pluripotent status,FTH1 stable knock-down ESCs were obtained using lentiviral vector plasmids. The effect of FTH1 silencing on hESCs pluripotency was evaluated through alkaline phosphatase (AP) staining, immunofluorescence and embryoid bodies (EBs) formation assay. Western blotting and qRT-PCR analysis were performed to assess the involvement of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and pentose phosphate pathway (PPP) in the antioxidant response. ROS levels and mitochondrial functionality were explored by flow cytometry. Seahorse Analyzer was used to evaluate metabolic and bioenergetic profiles. Results: Our findings clearly show that FTH1 silencing in hESCs does not correlate with increased ROS production nor with redox status strengthening the concept that hESCs are extremely resistant and, to certain extent, even refractory to the pattern of results produced in other cell lines. Collectively, our results demonstrate that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP) which crosstalk in driving hESCs antioxidant cascade events able to antagonize the effects of FTH1 silencing.Conclusion: to our knowledge, this is the first evidence of a crosstalk between FTH1 silencing and Nrf2 pathway activation in hESCs, casting a new light on how human ESCs perform under oxidative stress conditions. Our findings go beyond previous reports, showing how the Nrf2 pathway, in combination with PPP activation, regulates the molecular signature underlying ESCs defence mechanisms against oxidative stress mediated by FTH1 downregulation.

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Generation of iPSC lines from two patients affected by febrile seizure due to inherited missense mutation in SCN1A gene

Cited by 14 publications

References 1 publication

Direct Visualization and Identification of Membrane Voltage‐Gated Sodium Channels from Human iPSC‐Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy

Direct Visualization and Identification of Membrane Voltage‐Gated Sodium Channels from Human iPSC‐Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy

Uncovering the Metabolic and Stress Responses of Human Embryonic Stem Cells to FTH1 Gene Silencing

Dissecting the Molecular Response of Human Escs to Iron-Mediated Oxidative Stress by Genetic Silencing of FTH1 Gene

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