scholarly journals A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariana L. Casalia ◽  
Juan Cruz Casabona ◽  
Corina García ◽  
Verónica Cavaliere Candedo ◽  
Héctor Ramiro Quintá ◽  
...  
Keyword(s):  
Gene Expression ◽  
Action Potential ◽  
Genomic Analysis ◽  
Patient Specific ◽  
Childhood Epilepsy ◽  
Ph Domain ◽  
Neuronal Marker ◽  
Functional Genomic Analysis ◽  
Induced Pluripotent

Abstract Background Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. Methods Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. Results Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. Conclusions We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

scholarly journals Mitochondrial Abnormalities in Induced Pluripotent Stem Cells-Derived Motor Neurons from Patients with Riboflavin Transporter Deficiency

Antioxidants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1252
Author(s):  
Fiorella Colasuonno ◽  
Enrico Bertini ◽  
Marco Tartaglia ◽  
Claudia Compagnucci ◽  
Sandra Moreno
Keyword(s):  
Motor Neurons ◽  
Neurodegenerative Disorder ◽  
Ion Beam ◽  
Sensorineural Deafness ◽  
Microscopic Analysis ◽  
Patient Specific ◽  
Neuronal Marker ◽  
Transporter Deficiency ◽  
Induced Pluripotent

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and βIII-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs—emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants.

scholarly journals The Promise of Human Induced Pluripotent Stem Cells in Dental Research

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Thekkeparambil Chandrabose Srijaya ◽  
Padmaja Jayaprasad Pradeep ◽  
Rosnah Binti Zain ◽  
Sabri Musa ◽  
Noor Hayaty Abu Kasim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Patient Specific ◽  
Dental Research ◽  
Cell Based Therapy ◽  
Induced Pluripotent ◽  
Disease Specific

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC linesin vitrofrom patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

Abstract 17203: Exosomes From Induced Pluripotent Stem Cell-Derived Cardiomyocytes Salvage the Injured Myocardium by Modulation of Autophagy

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Michelle R Santoso ◽  
Yuko Tada ◽  
Gentaro Ikeda ◽  
Ji-Hye Jung ◽  
Evgeniya Vaskova ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Similar Efficacy ◽  
Major Constituent ◽  
Patient Specific ◽  
Parent Cell ◽  
Paracrine Factors ◽  
Intramyocardial Injection ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Background: Induced pluripotent stem cells (iPSCs) and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for myocardial injury (MI). Our previous work showed that the iCMs restore the injured murine myocardium through secretion of paracrine factors, modulating apoptotic pathways to restore the murine peri-infarct region (PIR). Hypothesis: iCM-derived exosomes (iCM-Ex), a major constituent of the iCM secretome, may salvage the injured cardiomyocytes in the PIR. Methods: iCM-Ex were precipitated from iCM supernatant and characterized using various molecular analyses. Immunodeficient mice sustained MIs and received iCMs, iCM-Ex, or PBS control via direct intramyocardial injection into the PIR. Cardiac MRI assessed LV ejection fraction (LVEF) and viability at 2- and 4-week post-injection. iCMs, iCM-Ex, and PIR tissue were isolated for molecular and histological analyses. Results: iCM-Ex measured approximately 142 nm and expressed CD63 and CD9. iCM and iCM-Ex miRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. In vitro iCM apoptosis was increased significantly by hypoxia and exosome biogenesis inhibition while iCM-Ex or rapamycin reduced iCM apoptosis (p<0.05, vs. control). Mice treated with iCMs or iCM-Ex had significantly improved LVEF and LV viability compared to the control (p<0.05). Apoptosis and fibrosis were significantly reduced in iCM- and iCM-Ex treated mice. Autophagy and associated mTOR signaling pathway were significantly enhanced in iCM-Ex treatment group. Conclusions: iCM-Ex demonstrated similar efficacy as the iCMs in improving post-MI cardiac function by regulating autophagy and apoptosis of hypoxia injured cardiomyocytes. This finding represents the potential of cell-free, patient-specific biologic to treat ischemic cardiomyopathy by stimulation of an endogenous repair mechanism.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

scholarly journals Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Audrey Chabrat ◽  
Emmanuelle Lacassagne ◽  
Rodolphe Billiras ◽  
Sophie Landron ◽  
Amélie Pontisso-Mahout ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Neurodegenerative Diseases ◽  
Dopaminergic Neurons ◽  
Adult Stem Cells ◽  
Morphological Changes ◽  
Direct Conversion ◽  
Patient Specific ◽  
Induced Pluripotent

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson’s disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

scholarly journals Human Cardiac Organoids for Modeling Genetic Cardiomyopathy

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1733 ◽  
Author(s):  
Michele Filippo Buono ◽  
Lisa von Boehmer ◽  
Jaan Strang ◽  
Simon P. Hoerstrup ◽  
Maximilian Y. Emmert ◽  
...  
Keyword(s):  
Cardiac Fibroblasts ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Promising Tool ◽  
Vitro Model ◽  
Human Cardiac Fibroblasts ◽  
Induced Pluripotent ◽  
First Time ◽  
Design And Testing

Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)—from healthy subjects and cardiomyopathy patients—human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids’ suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.

scholarly journals Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

2018 ◽  
Vol 115 (15) ◽  
pp. 3936-3941 ◽  
Author(s):  
Masayuki Hata ◽  
Hanako O. Ikeda ◽  
Sachiko Iwai ◽  
Yuto Iida ◽  
Norimoto Gotoh ◽  
...  
Keyword(s):  
Free Cholesterol ◽  
Cell Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Patient Specific ◽  
Rpe Cells ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Bietti's Crystalline Dystrophy

Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

scholarly journals HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Ji-Yon Kim ◽  
So-Youn Woo ◽  
Young Bin Hong ◽  
Heesun Choi ◽  
Jisoo Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Neuropathy ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Motor Neuropathy ◽  
Mitochondrial Movement ◽  
Induced Pluripotent

The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 703-703
Author(s):  
Naoya Takayama ◽  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Sou Nakamura ◽  
Sho-ichi Hirose ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Patient Specific ◽  
Gene Correction ◽  
Donor Skin ◽  
Induced Pluripotent

Abstract Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

Functional genomics of pollen tube–pistil interactions in Arabidopsis

2010 ◽  
Vol 38 (2) ◽  
pp. 593-597 ◽  
Author(s):  
Ravishankar Palanivelu ◽  
Mark A. Johnson
Keyword(s):  
Pollen Tube ◽  
Regulatory Networks ◽  
Genomic Analysis ◽  
Pollen Tubes ◽  
Cellular Growth ◽  
Functional Genomic ◽  
Reverse Genetic ◽  
Guidance Cues ◽  
Functional Genomic Analysis

The pollen tube represents an attractive model system for functional genomic analysis of the cell–cell interactions that mediate guided cellular growth. The pollen tube extends through pistil tissues and responds to guidance cues that direct the tube towards an ovule, where it releases sperm for fertilization. Pollen is readily isolated from anthers, where it is produced, and can be induced to produce a tube in vitro. Interestingly, pollen tube growth is significantly enhanced in pistils, and pollen tubes are rendered competent to respond to guidance cues after growth in a pistil. This potentiation of the pollen tube by the pistil suggested that pollen tubes alter their gene-expression programme in response to their environment. Recently, the transcriptomes of pollen tubes grown in vitro or through pistil tissues were determined. Significant changes in the transcriptome were found to accompany growth in vitro and through the pistil tissues. Reverse genetic analysis of pollen-tube-induced genes identified a new set of factors critical for pollen tube extension and navigation of the pistil environment. Recent advances reviewed in the present paper suggest that functional genomic analysis of pollen tubes has the potential to uncover the regulatory networks that shape the genetic architecture of the pollen tube as it responds to migratory cues produced by the pistil.

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