Littérature scientifique sur le sujet « IPSC-Derived neural models »

Abstract Background Traumatic brain injury (TBI) is a major global health problem with limited treatment options. Induced pluripotent stem cells (iPSCs) have emerged as a promising therapy for neural regeneration and repair after TBI. Aims & Objectives This systematic review aimed to evaluate preclinical studies on the efficacy of iPSC- based therapies for functional recovery after TBI. Method From, PubMed, Ovid Medline, Cochrane Library, SCOPUS, and Web of Science 102 studies were found for studies on iPSCs in TBI animal models. Included studies (n=9) used neural stem cells derived from iPSCs transplanted into rodent models of TBI. Outcome measures were brain injury volume, functional recovery, and tissue repair biomarkers. Study quality was assessed using a 10-point scale. Results The majority of studies showed significant improvement in motor function, cognition, and social behavior with iPSC therapy. Transplanted iPSC-neural stem cells migrated to injury sites, differentiated into neurons and glia, reduced lesion size, and increased neural repair markers. Higher quality scores were noted for studies reporting randomization, blinded assessment, and temperature control. Discussion & Conclusions Preclinical evidence demonstrates the potential of iPSC-derived neural stem cell transplantation to improve functional outcomes after TBI through neuroregenerative effects. Further research is warranted to evaluate safety, optimize protocols, and translate findings to clinical trials for TBI patients. iPSC-based therapies may offer new hope for recovery after this devastating injury.

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

The establishment of human-induced pluripotent stem cell (iPSC) models from sporadic Alzheimer’s disease (sAD) patients is necessary and could potentially benefit research into disease etiology and therapeutic strategies. However, the development of sAD iPSC models is still limited due to the multifactorial nature of the disease. Here, we extracted peripheral blood mononuclear cells (PBMCs) from a patient with sAD and induced them into iPSC by introducing the Sendai virus expressing Oct3/4, Sox2, c-Myc, and Klf4, which were subsequently induced into neural cells to build the cell model of AD. Using alkaline phosphatase staining, immunofluorescence staining, karyotype analysis, reverse transcription-polymerase chain reaction (RT-PCR), and teratoma formation in vitro, we demonstrated that the iPSC derived from PMBCs (PBMC-iPSC) had a normal karyotype and potential to differentiate into three embryonic layers. Immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) suggested that PBMC-iPSCs were successfully differentiated into neural cells. Detection of beta-amyloid protein oligomer (AβO), beta-amyloid protein 1-40 (Aβ 1-40), and beta-amyloid protein 1-42 (Aβ 1-42) indicated that the AD cell model was satisfactorily constructed in vitro. In conclusion, this study has successfully generated an AD cell model with pathological features of beta-amyloid peptide deposition using PBMC from a patient with sAD.

AbstractDuring the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer’s disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery. Graphical abstract

In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson’s disease, and Alzheimer’s disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.

Unexpected toxicity in areas such as cardiotoxicity, hepatotoxicity, and neurotoxicity is a serious complication of clinical therapy and one of the key causes for failure of promising drug candidates in development. Animal studies have been widely used for toxicology research to provide preclinical security evaluation of various therapeutic agents under development. Species differences in drug penetration of the blood–brain barrier, drug metabolism, and related toxicity contribute to failure of drug trials from animal models to human. The existing system for drug discovery has relied on immortalized cell lines, animal models of human disease, and clinical trials in humans. Moreover, drug candidates that are passed as being safe in the preclinical stage often show toxic effects during the clinical stage. Only around 16% drugs are approved for human use. Research on induced pluripotent stem cells (iPSCs) promises to enhance drug discovery and development by providing simple, reproducible, and economically effective tools for drug toxicity screening under development and, on the other hand, for studying the disease mechanism and pathways. In this review, we provide an overview of basic information about iPSCs, and discuss efforts aimed at the use of iPSC-derived hepatocytes, cardiomyocytes, and neural cells in drug discovery and toxicity testing.

Epigenetic mechanisms play a role in human disease but their involvement in pathologies from the central nervous system has been hampered by the complexity of the brain together with its unique cellular architecture and diversity. Until recently, disease targeted neural types were only available as postmortem materials after many years of disease evolution. Current in vitro systems of induced pluripotent stem cells (iPSCs) generated by cell reprogramming of somatic cells from patients have provided valuable disease models recapitulating key pathological molecular events. Yet whether cell reprogramming on itself implies a truly epigenetic reprogramming, the epigenetic mechanisms governing this process are only partially understood. Moreover, elucidating epigenetic regulation using patient-specific iPSC-derived neural models is expected to have a great impact to unravel the pathophysiology of neurodegenerative diseases and to hopefully expand future therapeutic possibilities. Here we will critically review current knowledge of epigenetic involvement in neurodegenerative disorders focusing on the potential of iPSCs as a promising tool for epigenetic research of these diseases.

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Les troubles neurodéveloppementaux (TND) touchent environ 10 % des enfants et constituent une source majeure d'invalidité tout au long de la vie. Caractérisés par un développement défectueux du cerveau et une grande variabilité du tableau clinique des patients, qui compromet le diagnostic et l'émergence de solutions thérapeutiques, ils représentent un coût humain, sociétal et économique important. L'objectif de ce projet est de mieux comprendre une caractéristique commune des TND - la dérégulation des voies de réponse au stress - qui constituerait une clef de lecture pour comprendre ces pathologies. L'intégration des processus déclenchés par le stress est régie par les facteurs de transcription du choc thermique (HSF), qui sont fortement dérégulés dans plusieurs TND. Cela a deux conséquences : une altération de la réponse au stress des cellules neurales qui entraîne des défauts dans le développement du cerveau. Nous avons participé a montré que ces HSF sont essentiels au bon développement du cerveau. Plus précisément, l'équipe a démontré que HSF2 joue un rôle clef dans la régulation de la prolifération des cellules progénitrices et la migration neuronale dans le cortex en modulant l'expression de gène impliqués dans l'adhésion cellulaire. La modulation pharmacologique de cette voie pourrait donc offrir de nouvelles possibilités thérapeutiques. Dans une première étude, les mécanismes sous-jacents à la dérégulation des HSF ont été étudiés dans les cellules de patients atteints du syndrome de Rubinstein-Taybi (RSTS), un TND rare d'origine génétique causé par des mutations dans les gènes CREBBP ou EP300. Notre étude a montré une diminution des niveaux protéiques de HSF2 dans les fibroblastes et dans les modèles neuraux (2D et 3D) dérivés à partir de cellules souches pluripotentes induites (iPSC) provenant de patients RSTS. Cette diminution des niveaux protéiques de HSF2 résultait d'un défaut d'acétylation par CBP ou EP300, conduisant à l'ubiquitination et à la dégradation par le protéasome. En conséquence, les cellules RSTS présentaient une altération de la réponse au stress et une réduction de l'expression de gènes essentiels au développement neural, en particulier la N-cadhérine. La restauration des niveaux de HSF2, soit par l'inhibition du protéasome, soit par des mutations imitant l'acétylation, a permis de rétablir à la fois la réponse au stress et l'expression des gènes du neurodéveloppement. Nous avons constaté que la perturbation de la voie CBP/EP300-HSF2-N-cadhérine est récapitulée dans les modèles neuraux RSTS, qui présentent des anomalies de prolifération liées à une altération de l'adhésion cellule-cellule, en particulier dans la voie de la N-cadhérine. Sur la base de ces résultats et en collaboration avec Ksilink, mon projet de thèse CIFRE vise à développer un modèle cellulaire de TND basé sur les patients RSTS. Ce modèle permettra d'explorer comment les perturbations de la voie HSF pourraient contribuer à divers TND. Pour atteindre cet objectif, j'ai d'abord généré un mutant HSF2 qui mime la forme acétylée de la protéine dans les iPSC dérivés de fibroblastes de patients RSTS. En utilisant ce modèle isogénique comme référence, j'ai développé et validé un modèle de culture neural bidimensionnel et identifié de nouvelles cibles et phénotypes dépendants de HSF2 via une approche multiparamétrique allant de la transcriptomique à haut débit à des analyses morphologiques des cellules. Cette approche a permis d'identifier le facteur pro neuronal, ASCL1, et un phénotype morphologique, la formation de rosettes, comme clefs de lecture pour l'analyse par imagerie à haut contenu. Sur la base de ces deux phénotypes, j'ai utilisé le modèle neural pour cribler une sélection de molécules à potentiel thérapeutique par imagerie à haut contenu. Ces travaux ouvriront la voie à de nouvelles approches thérapeutiques visant à moduler les voies de réponse au stress, offrant ainsi de nouvelles possibilités de traitement des TND
Neurodevelopmental disorders (NDD) affect around 10% of children and are a major source of lifelong disability. Characterised by defective brain development and great variability in the clinical picture of patients, which compromises diagnosis and the emergence of therapeutic solutions, they represent a significant human, societal and economic cost. The aim of this project is to gain a better understanding of a common feature of NDDs - the deregulation of stress response pathways - which could provide a readout to understanding these pathologies. The integration of processes triggered by stress is governed by heat shock transcription factors (HSFs), which are strongly deregulated in several NDDs. This has two consequences: an altered stress response in neural cells leading to defects in brain development. We have helped to show that these HSFs are essential for proper brain development. More specifically, the team demonstrated that HSF2 plays a key role in regulating the proliferation of progenitor cells and neuronal migration in the cortex by modulating the expression of genes involved in cell adhesion. Pharmacological modulation of this pathway could therefore offer new therapeutic possibilities. In a first study, the mechanisms underlying HSF deregulation were investigated in cells from patients with Rubinstein-Taybi syndrome (RSTS), a rare genetic NDD caused by mutations in the CREBBP or EP300 genes. Our study showed a decrease in HSF2 protein levels in fibroblasts and in neural models (2D and 3D) derived from induced pluripotent stem cells (iPSCs) from RSTS patients. This decrease in HSF2 protein levels resulted from a defect in acetylation by CBP or EP300, leading to ubiquitination and degradation by the proteasome. As a result, RSTS cells showed an altered stress response and reduced expression of genes essential for neural development, in particular N-cadherin. Restoration of HSF2 levels, either by proteasome inhibition or by acetylation-mimicking mutations, restored both the stress response and the expression of neurodevelopmental genes. We found that disruption of the CBP/EP300-HSF2-N-cadherin pathway is recapitulated in RSTS neural models, which display proliferation abnormalities linked to altered cell-cell adhesion, particularly in the N-cadherin pathway. On the basis of these results and in collaboration with Ksilink, my CIFRE thesis project aims to develop a cellular model of NDD based on RSTS patients. This model will enable us to explore how perturbations in the HSF pathway could contribute to various NDDs. To achieve this objective, I first generated an HSF2 mutant that mimics the acetylated form of the protein in iPSCs derived from RSTS patient fibroblasts. Using this isogenic model as a reference, I developed and validated a two-dimensional neural culture model and identified new HSF2-dependent targets and phenotypes using a multiparametric approach ranging from high-throughput transcriptomics to cell morphological analyses. This approach made it possible to identify the pro-neuronal factor, ASCL1, and a morphological phenotype, rosette formation, as key readouts for analysis by high-content imaging. On the basis of these two phenotypes, I used the neural model to screen a selection of molecules with therapeutic potential using high-content imaging. This work will pave the way for new therapeutic approaches aimed at modulating stress response pathways, thereby opening up new possibilities for the treatment of NDD

Mouse and cellular models of ALS including stem cells have revealed tremendous insight into the molecular processes that lead to ALS. Models of ALS and other neurodegenerative diseases have led to emergent molecular themes that span several diseases. Future models must account for neuronal subtype specificity of different neurodegenerative diseases, particularly between tightly related diseases such as FTD and ALS. Human iPSC-derived motor neurons offer promise both with regard to the use of human cells and in particular the ability to model sporadic disease, which is critically important given the overwhelming abundance of sporadic disease in ALS and other neurodegenerative diseases.

AbstractImmature cardiomyocytes, such as those obtained by stem cell differentiation, have been shown to be useful alternatives to mature cardiomyocytes, which are limited in availability and difficult to obtain, for evaluating the behaviour of drugs for treating arrhythmia. In silico models of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can be used to simulate the behaviour of the transmembrane potential and cytosolic calcium under drug-treated conditions. Simulating the change in action potentials due to various ionic current blocks enables the approximation of drug behaviour. We used eight machine learning classification models to predict partial block of seven possible ion currents $$ (\textit{I}_{\textit{CaL}},\textit{I}_{\textit{Kr}},\textit{I}_{\textit{to}},\textit{I}_{\textit{K1}},\textit{I}_{\textit{Na}},\textit{I}_{\textit{NaL}} and \textit{I}_{\textit{Ks}}) $$ in a simulated dataset containing nearly 4600 action potentials represented as a paired measure of transmembrane potential and cytosolic calcium. Each action potential was generated under 1 $$ \textit{H}_{\textit{z}} $$ pacing. The Convolutional Neural Network outperformed the other models with an average accuracy of predicting partial ionic current block of 93% in noise-free data and 72% accuracy with 3% added random noise. Our results show that $$ \textit{I}_{\textit{CaL}} $$ and $$ \textit{I}_{\textit{Kr}} $$ current block were classified with high accuracy with and without noise. The classification of $$ \textit{I}_{\textit{to}} $$ , $$ \textit{I}_{\textit{K1}} $$ and $$ \textit{I}_{\textit{Na}} $$ current block showed high accuracy at 0% noise, but showed a significant decrease in accuracy when noise was added. Finally, the accuracy of $$ \textit{I}_{\textit{NaL}} $$ and $$ \textit{I}_{\textit{Ks}} $$ classification were relatively lower than the other current blocks at 0% noise and also showed a significant drop in accuracy when noise was added. In conclusion, these machine learning methods may present a pathway for estimating drug response in adult phenotype cardiac systems, but the data must be sufficiently filtered to remove noise before being used with classifier algorithms.

Abstract Advances in human induced pluripotent stem cell (iPSC) approaches have greatly expanded the use of patient-derived cellular models, including cortical-like neurons and brain organoids, to study genetic epilepsies. New protocols to differentiate iPSCs into various neural cell types, and to generate brain region-specific organoids, have accelerated progress. In addition, the application of gene editing techniques adds rigor to these studies and offers the opportunity to model rare genetic epilepsies by enabling correction or insertion of mutations to generate isogenic controls or virtual patients, respectively. Studies using patient-derived cells have provided insight into the mechanisms underlying seizures and associated comorbidities for an increasing number of epilepsy gene variants. In this chapter, methods for generating iPSCs, the culture and gene editing of human pluripotent stem cells (hPSCs; both iPSCs and human embryonic stem cells), and differentiation strategies to derive cortical-like neurons are described. Next, physiological assays that can be applied to study hPSC-derived neuronal and network activity are discussed. Finally, the chapter reviews studies interrogating the functional effects of specific epilepsy gene variants using hPSC-derived 2D neural and 3D brain organoid cultures, as well as the use of iPSC-derived cardiac myocytes to study the most devastating epilepsy complication, sudden unexpected death in epilepsy (SUDEP).The chapter ends with a description of current challenges and future directions for hPSC modeling of genetic epilepsies.

Background: Parkinson’s disease (PD) is a neurological disorder that affects movement, mainly due to damage and degeneration of the nigrostriatal dopaminergic pathway. The diagnosis is made through a clinical neurological analysis where motor characteristics are considered. There is still no cure, and treatment strategies are focused on symptoms control. Cell replacement therapies emerge as an alternative. Objective: This review focused on current techniques of induced pluripotent stem cells (iPSCs). Methods: The search terms used were: “Parkinson’s Disease”, “Stem cells” and “iPSC”. Open articles written in English, from 2016-21 were selected in the Pubmed database, 10 publications were identified. Results: With the modernization of iPSC, it was possible to reprogram pluripotent human somatic cells and generate dopaminergic neurons and individual-specific glial cells. To understand the molecular basis, cell and animal models of neurons and organelles are currently being employed. Organoids are derived from stem cells in a three-dimensional matrix, such as matrigel or hydrogels derived from animals. The neuronal models are: α-synuclein (SNCA), leucine-rich repeat kinase2 (LRRK2), PARK2, putative kinase1 induced by phosphatase and tensin homolog (PINK1), DJ-1. Both models offer opportunities to investigate pathogenic mechanisms of PD and test compounds on human neurons. Conclusions: Cell replacement therapy is promising and has great capacity for the treatment of neurodegenerative diseases. Studies using iPSC neuron and PD organoid modeling is highly valuable in elucidating relevants neuronal pathways and therapeutic targets, moreover providing important models for testing future therapies.

Progressive Supranuclear Palsy (PSP) is a neurodegenerative tauopathy and, to date, the pathophysiological mechanisms in PSP that lead to Tau hyperphosphorylation and neurodegeneration are not clear. The development of a model using neural cell lines derived from patients has the potential to identify molecules and possible biomarkers. We developed a model of induced pluripotent stem cells iPSC-derived astrocytes to investigate the pathophysiology of PSP, particularly early events that might contribute to Tau hyperphosphorylation, applying an omics approach to detect differentially expressed genes, metabolites, and proteins, including those from the secretome. Skin fibroblasts from PSP patients and controls were reprogrammed to iPSCs, which were further differentiated into neuroprogenitor cells and astrocytes. In the 5th passage, astrocytes were harvested for total ribonucleic acid sequencing. Intracellular and secreted proteins were processed for proteomics experiments. Metabolomics profiling was obtained from supernatants only. We identified hundreds of differentially expressed genes in PSP. The main networks were related to cell cycle activation. Several proteins were found exclusively secreted by the PSP group. The cellular processes related to the cell cycle and mitotic proteins, chaperonins of the TriC/CCT pathway, and redox signaling are enriched in the secretome of the PSP group. Moreover, we found spatial segregation by PCA in the metabolomics data, indicating distinct sets of metabolites between PSP and control groups. Our iPSC-derived astrocyte model can provide distinct molecular signatures for PSP patients and it is useful to elucidate the initial stages of PSP pathogenesis.