Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise - even asymptomatically - is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to healthy control cells by a comprehensive multi-omics analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrate that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers. Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development

Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise - even asymptomatically - is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to healthy control cells by a comprehensive multi-omics analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrate that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers. Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development

Myogenesis modelled by human pluripotent stem cells: a multi‐omic study of Duchenne myopathy early onset

International audienceBackground Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. Methods We have used both human tissue-derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi-omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR-edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC-derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC-derived myotubes. Results Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps-mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle-dmd.omics.ovh/. Conclusions Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome

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Cell Therapy for skin replacement : Study of the molecular mechanisms involved in the production of human skin reconstructed from pluripotent stem cell.

Ces dernières années, les cellules souches pluripotentes humaines ont suscité un immense intérêt dans le domaine de la thérapie cellulaire et de la médecine régénérative. Cependant, le défi majeur pour la translation clinique de ces thérapies réside dans l'établissement de contrôles qualité standardisés afin de surveiller efficacement la production. Les technologies "OMICS", qui englobent diverses analyses à grande échelle, offrent la possibilité d'identifier les acteurs clés et les voies moléculaires impliquées dans des processus dynamiques tels que la production cellulaire. Ces biomarqueurs peuvent ensuite être utilisés pour qualifier et contrôler le succès de la production cellulaire. Ainsi, au cours de cette thèse, l'objectif était de concevoir des méthodologies s'appuyant sur ces approches pour caractériser au niveau moléculaire des modèles cellulaires destinés à des applications cliniques. L'objectif final étant d'établir des contrôles qualité capables d'anticiper et de gérer les variations et les déviations, réduisant ainsi les coûts et garantissant la réussite du processus de différenciation. Pour valider cette approche, nous avons centré notre étude sur la différenciation de cellules pluripotentes en cellules de la peau, telles que les mélanocytes et les kératinocytes basaux. Ces processus sont actuellement évalués à l'Institut I-Stem pour la production cellulaire clinique destinée au traitement des atteintes cutanées.Dans ce contexte, le premier objectif de ma thèse visait à caractériser au niveau génomique et transcriptomique le protocole de différenciation des cellules pluripotentes humaines en mélanocytes. Ces analyses ont permis d'évaluer la stabilité génomique lors de l'expansion des mélanocytes et de développer un test adaptable à différents modèles cellulaires. Les analyses transcriptomiques ont révélé des gènes clés associés à ce processus, ouvrant ainsi la voie à leur évaluation et à leur suivi pour la qualification de la différenciation.Le deuxième objectif de cette étude était d'utiliser des analyses de séquençage de cellules uniques pour explorer l'hétérogénéité cellulaire de la différenciation des kératinocytes basaux dérivés de cellules pluripotentes humaines. Ces données ont été combinées avec des approches protéomiques afin d'identifier les acteurs clés de ce processus cellulaire. À la suite de ces analyses, nous nous sommes concentrés sur la modulation des protéines sécrétées dans le milieu de culture. Cette approche nous a permis d'identifier des marqueurs pertinents qui reflètent avec précision l'évolution de la production directement dans le surnageant cellulaire. Ces résultats offrent désormais la possibilité de mettre en place des contrôles qualité non invasifs pour la production de cellules dérivées de cellules pluripotentes humaines.In recent years, human pluripotent stem cells have emerged as a promising tool for cellular therapy and regenerative medicine. However, a major challenge for the clinical translation of these therapies lies in the establishement of standardized quality controls to effectively monitor productions. "OMICS" technologies, encompassing various large-scale analyses, offer the potential to identify key players and molecular pathways involved in dynamic processes such as cell production. These biomarkers can then be used to qualify, control, and monitor cellular production. In this context, the main objective of this thesis was to develop methodologies based on these approaches to characterize at molecular level, cell models intended for clinical applications. The ultimate goal was to establish quality controls capable of anticipating and managing variations and deviations, reducing costs and ensuring the success of the differentiation process. To validate this approach, our study focused on the differentiation of pluripotent cells into skin cells such as melanocytes and basal keratinocytes. These processes are currently evaluated at the I-Stem Institute for clinical cell production for the treatment of skin disorders.In this context, the first objective of my thesis aimed to characterize at genomic and transcriptomic scale the differentiation of human pluripotent cells into melanocytes. These analyses allow to assess genomic stability during melanocyte expansion and to develop an adaptable test for various cell models. Transcriptomic analyses revealed key genes associated with this process, paving the way for their evaluation and monitoring for differentiation qualification.The second objective of this study was to use single-cell sequencing analyses to explore the cellular heterogeneity of keratinocyte differentiation derived from human pluripotent cells. These data were combined with proteomic approaches to identify key players of this cellular process. Following these analyses, we focused on the modulation of proteins secreted in the culture medium. This approach allowed to identify relevant markers that accurately reflect the evolution of production directly in the cellular supernatant. These results now provide the opportunity to establish non-invasive quality controls for the production of cells derived from human pluripotent cells

Thérapie cellulaire de la peau : Étude des mécanismes moléculaires impliqués dans les procédés de production de peau humaine reconstituée à partir de dérivés de cellules souches pluripotentes

In recent years, human pluripotent stem cells have emerged as a promising tool for cellular therapy and regenerative medicine. However, a major challenge for the clinical translation of these therapies lies in the establishement of standardized quality controls to effectively monitor productions. "OMICS" technologies, encompassing various large-scale analyses, offer the potential to identify key players and molecular pathways involved in dynamic processes such as cell production. These biomarkers can then be used to qualify, control, and monitor cellular production. In this context, the main objective of this thesis was to develop methodologies based on these approaches to characterize at molecular level, cell models intended for clinical applications. The ultimate goal was to establish quality controls capable of anticipating and managing variations and deviations, reducing costs and ensuring the success of the differentiation process. To validate this approach, our study focused on the differentiation of pluripotent cells into skin cells such as melanocytes and basal keratinocytes. These processes are currently evaluated at the I-Stem Institute for clinical cell production for the treatment of skin disorders.In this context, the first objective of my thesis aimed to characterize at genomic and transcriptomic scale the differentiation of human pluripotent cells into melanocytes. These analyses allow to assess genomic stability during melanocyte expansion and to develop an adaptable test for various cell models. Transcriptomic analyses revealed key genes associated with this process, paving the way for their evaluation and monitoring for differentiation qualification.The second objective of this study was to use single-cell sequencing analyses to explore the cellular heterogeneity of keratinocyte differentiation derived from human pluripotent cells. These data were combined with proteomic approaches to identify key players of this cellular process. Following these analyses, we focused on the modulation of proteins secreted in the culture medium. This approach allowed to identify relevant markers that accurately reflect the evolution of production directly in the cellular supernatant. These results now provide the opportunity to establish non-invasive quality controls for the production of cells derived from human pluripotent cells.Ces dernières années, les cellules souches pluripotentes humaines ont suscité un immense intérêt dans le domaine de la thérapie cellulaire et de la médecine régénérative. Cependant, le défi majeur pour la translation clinique de ces thérapies réside dans l'établissement de contrôles qualité standardisés afin de surveiller efficacement la production. Les technologies "OMICS", qui englobent diverses analyses à grande échelle, offrent la possibilité d'identifier les acteurs clés et les voies moléculaires impliquées dans des processus dynamiques tels que la production cellulaire. Ces biomarqueurs peuvent ensuite être utilisés pour qualifier et contrôler le succès de la production cellulaire. Ainsi, au cours de cette thèse, l'objectif était de concevoir des méthodologies s'appuyant sur ces approches pour caractériser au niveau moléculaire des modèles cellulaires destinés à des applications cliniques. L'objectif final étant d'établir des contrôles qualité capables d'anticiper et de gérer les variations et les déviations, réduisant ainsi les coûts et garantissant la réussite du processus de différenciation. Pour valider cette approche, nous avons centré notre étude sur la différenciation de cellules pluripotentes en cellules de la peau, telles que les mélanocytes et les kératinocytes basaux. Ces processus sont actuellement évalués à l'Institut I-Stem pour la production cellulaire clinique destinée au traitement des atteintes cutanées.Dans ce contexte, le premier objectif de ma thèse visait à caractériser au niveau génomique et transcriptomique le protocole de différenciation des cellules pluripotentes humaines en mélanocytes. Ces analyses ont permis d'évaluer la stabilité génomique lors de l'expansion des mélanocytes et de développer un test adaptable à différents modèles cellulaires. Les analyses transcriptomiques ont révélé des gènes clés associés à ce processus, ouvrant ainsi la voie à leur évaluation et à leur suivi pour la qualification de la différenciation.Le deuxième objectif de cette étude était d'utiliser des analyses de séquençage de cellules uniques pour explorer l'hétérogénéité cellulaire de la différenciation des kératinocytes basaux dérivés de cellules pluripotentes humaines. Ces données ont été combinées avec des approches protéomiques afin d'identifier les acteurs clés de ce processus cellulaire. À la suite de ces analyses, nous nous sommes concentrés sur la modulation des protéines sécrétées dans le milieu de culture. Cette approche nous a permis d'identifier des marqueurs pertinents qui reflètent avec précision l'évolution de la production directement dans le surnageant cellulaire. Ces résultats offrent désormais la possibilité de mettre en place des contrôles qualité non invasifs pour la production de cellules dérivées de cellules pluripotentes humaines

Thérapie cellulaire de la peau : Étude des mécanismes moléculaires impliqués dans les procédés de production de peau humaine reconstituée à partir de dérivés de cellules souches pluripotentes

In recent years, human pluripotent stem cells have emerged as a promising tool for cellular therapy and regenerative medicine. However, a major challenge for the clinical translation of these therapies lies in the establishement of standardized quality controls to effectively monitor productions. "OMICS" technologies, encompassing various large-scale analyses, offer the potential to identify key players and molecular pathways involved in dynamic processes such as cell production. These biomarkers can then be used to qualify, control, and monitor cellular production. In this context, the main objective of this thesis was to develop methodologies based on these approaches to characterize at molecular level, cell models intended for clinical applications. The ultimate goal was to establish quality controls capable of anticipating and managing variations and deviations, reducing costs and ensuring the success of the differentiation process. To validate this approach, our study focused on the differentiation of pluripotent cells into skin cells such as melanocytes and basal keratinocytes. These processes are currently evaluated at the I-Stem Institute for clinical cell production for the treatment of skin disorders.In this context, the first objective of my thesis aimed to characterize at genomic and transcriptomic scale the differentiation of human pluripotent cells into melanocytes. These analyses allow to assess genomic stability during melanocyte expansion and to develop an adaptable test for various cell models. Transcriptomic analyses revealed key genes associated with this process, paving the way for their evaluation and monitoring for differentiation qualification.The second objective of this study was to use single-cell sequencing analyses to explore the cellular heterogeneity of keratinocyte differentiation derived from human pluripotent cells. These data were combined with proteomic approaches to identify key players of this cellular process. Following these analyses, we focused on the modulation of proteins secreted in the culture medium. This approach allowed to identify relevant markers that accurately reflect the evolution of production directly in the cellular supernatant. These results now provide the opportunity to establish non-invasive quality controls for the production of cells derived from human pluripotent cells.Ces dernières années, les cellules souches pluripotentes humaines ont suscité un immense intérêt dans le domaine de la thérapie cellulaire et de la médecine régénérative. Cependant, le défi majeur pour la translation clinique de ces thérapies réside dans l'établissement de contrôles qualité standardisés afin de surveiller efficacement la production. Les technologies "OMICS", qui englobent diverses analyses à grande échelle, offrent la possibilité d'identifier les acteurs clés et les voies moléculaires impliquées dans des processus dynamiques tels que la production cellulaire. Ces biomarqueurs peuvent ensuite être utilisés pour qualifier et contrôler le succès de la production cellulaire. Ainsi, au cours de cette thèse, l'objectif était de concevoir des méthodologies s'appuyant sur ces approches pour caractériser au niveau moléculaire des modèles cellulaires destinés à des applications cliniques. L'objectif final étant d'établir des contrôles qualité capables d'anticiper et de gérer les variations et les déviations, réduisant ainsi les coûts et garantissant la réussite du processus de différenciation. Pour valider cette approche, nous avons centré notre étude sur la différenciation de cellules pluripotentes en cellules de la peau, telles que les mélanocytes et les kératinocytes basaux. Ces processus sont actuellement évalués à l'Institut I-Stem pour la production cellulaire clinique destinée au traitement des atteintes cutanées.Dans ce contexte, le premier objectif de ma thèse visait à caractériser au niveau génomique et transcriptomique le protocole de différenciation des cellules pluripotentes humaines en mélanocytes. Ces analyses ont permis d'évaluer la stabilité génomique lors de l'expansion des mélanocytes et de développer un test adaptable à différents modèles cellulaires. Les analyses transcriptomiques ont révélé des gènes clés associés à ce processus, ouvrant ainsi la voie à leur évaluation et à leur suivi pour la qualification de la différenciation.Le deuxième objectif de cette étude était d'utiliser des analyses de séquençage de cellules uniques pour explorer l'hétérogénéité cellulaire de la différenciation des kératinocytes basaux dérivés de cellules pluripotentes humaines. Ces données ont été combinées avec des approches protéomiques afin d'identifier les acteurs clés de ce processus cellulaire. À la suite de ces analyses, nous nous sommes concentrés sur la modulation des protéines sécrétées dans le milieu de culture. Cette approche nous a permis d'identifier des marqueurs pertinents qui reflètent avec précision l'évolution de la production directement dans le surnageant cellulaire. Ces résultats offrent désormais la possibilité de mettre en place des contrôles qualité non invasifs pour la production de cellules dérivées de cellules pluripotentes humaines

SISTEMA: A large and standardized collection of transcriptome data sets for human pluripotent stem cell research

International audienceHuman pluripotent stem cells have ushered in an exciting new era for disease modeling, drug discovery, and cell therapy development. Continued progress toward realizing the potential of human pluripotent stem cells will be facilitated by robust data sets and complementary resources that are easily accessed and interrogated by the stem cell community. In this context, we present SISTEMA, a quality-controlled curated gene expression database, built on a valuable catalog of human pluripotent stem cell lines, and their derivatives for which transcriptomic analyses have been generated using a single experimental pipeline. SISTEMA functions as a one-step resource that will assist the stem cell community to easily evaluate the expression level for genes of interest, while comparing them across different hPSC lines, cell types, pathological conditions, or after pharmacological treatments

The Physical Economy of France (1830–2015). The History of a Parasite?

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