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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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

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein

International audienceAbstract Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides—besides transcription—an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP—together with the RBM4 splicing factor—in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X

Human iPSC-derived neurons reveal early developmental alteration of neurite outgrowth in the late-occurring neurodegenerative Wolfram syndrome

International audienceRecent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response—which is activated in late Wolfram degenerative processes—was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis

International audienceAlternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis

International audienceAlternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins