Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19-21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery

Generation of the first human in vitro model for McArdle disease based on iPSC Technology

McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.This work has been funded by grants from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (ISCIII): PI15/00484, CP16/00046 and PI18/00151 to MEG and PI17/02052 to JA (co-funded by European Regional Development Fund “A way to make Europe”); PI21/00162 and CPII21/00011 co-funded by the European Union to MEG. MdCOC receives grant support from the ‘Ministerio de Educación, Cultura y Deporte’ (FPU16/03895), ‘Fundación para la Investigación Biomédica Hospital 12 de Octubre’ (2022/0065, i+12-AY20220114-1) and EMBO Grant 8917. CL and MD were recipient of a fellowship from the French Ministry of Education. The work in FM’s laboratory was funded by “Association Française contre les Myopathies” (AFM; TRIM-RD and MoThARD) and from the Excellence Initiative of Aix-Marseille University-A*Midex, a French “investissement d’avenir programme” AMX-19-IET-007 through the Marseille Maladies Rares (MarMaRa) Institute (phD fellowship to CL)

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity

Bring It to an End: Does Telomeres Size Matter?

International audienceTelomeres are unique nucleoprotein structures. Found at the edge of each chromosome, their main purpose is to mask DNA ends from the DNA-repair machinery by formation of protective loops. Through life and cell divisions, telomeres shorten and bring cells closer to either cell proliferation crisis or senescence. Beyond this mitotic clock role attributed to the need for telomere to be maintained over a critical length, the very tip of our DNA has been shown to impact transcription by position effect. TPE and a long-reach counterpart, TPE-OLD, are mechanisms recently described in human biology. Still in infancy, the mechanism of action of these processes and their respective genome wide impact remain to be resolved. In this review, we will discuss recent findings on telomere dynamics, TPE, TPE-OLD, and lessons learnt from model organisms

I.09 Induced pluripotent stem cells for modeling neuromuscular disorders: development of disease-specific assays, live cells functional testing and drug design

International audienceInduced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting

Analysis of the 4q35 chromatin organization reveals distinct long-range interactions in patients affected with Facio-Scapulo-Humeral Dystrophy.

International audience1 Facio-Scapulo Humeral dystrophy (FSHD) is the third most common myopathy, affecting 1 amongst 10,000 individuals (FSHD1, OMIM #158900). This autosomal dominant pathology is associated in 95% of cases with genetic and epigenetic alterations in the subtelomeric region at the extremity of the long arm of chromosome 4 (q arm). A large proportion of the remaining 5% of cases carry a mutation in the SMCHD1 gene (FSHD2, OMIM #158901). Here, we explored the 3D organization of the 4q35 locus by three-dimensions DNA in situ fluorescent hybridization (3D-FISH) in primary fibroblasts isolated from patients and healthy donors. We found that D4Z4 contractions and/or SMCHD1 mutations impact the spatial organization of the 4q35 region and trigger changes in the expression of different genes. Changes in gene expression were corroborated in muscle biopsies suggesting that the modified chromatin landscape impelled a modulation in the level of expression of a number of genes across the 4q35 locus in FSHD. Using induced pluripotent stem cells (hIPSC), we further examined whether chromatin organization is inherited after reprogramming or acquired during differentiation and showed that folding of the 4q35 region is modified upon differentiation. These results together with previous findings highlight the role of the D4Z4 macrosatellite repeat in the topological organization of chromatin and further indicate that the D4Z4-dependent 3D structure induces transcriptional changes of 4q35 genes expression

SMCHD1 variants may induce variegated expression in Facio Scapulo Humeral Dystophy and Bosma Arhinia and microphtalmia syndrome

Abstract An expanding number of genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Recently, distinct genetic syndromes have been linked to mutations in the SMCHD1 gene. However, the function of this non-canonical SMC protein remains partly defined in Human tissues. To address this question, we determined its epi-signature in type 2 Facio Scapulo Humeral Dystrophy (FSHD2) and Bosma Arhinia and Microphtalmia Syndrome (BAMS) linked to heterozygous mutations in this gene. By combining RNA-Seq, DNA methylation profiling and ChIP-Seq, we showed that SMCHD1 regulates repressed chromatin but also cis -regulatory elements and enhancers. Our results emphasize dual functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable outcomes and targets depending on tissues. We propose that altered DNA methylation and long-range chromatin organization at a number of loci required for development and tissue differentiation, trigger variegated gene expression in rare genetic diseases linked to heterozygous SMCHD1 mutations

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism

International audienceObjective To investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulo-humeral dystrophy (FSHD). Methods By adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls. Results Using hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing-based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously. Conclusions Altogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macro-satellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism

In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype

International audienceMany genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation

AKT Signaling Modifies the Balance between Cell Proliferation and Migration in Neural Crest Cells from Patients Affected with Bosma Arhinia and Microphthalmia Syndrome

International audienceOver the recent years, the SMCHD1 (Structural Maintenance of Chromosome flexible Hinge Domain Containing 1) chromatin-associated factor has triggered increasing interest after the identification of variants in three rare and unrelated diseases, type 2 Facio Scapulo Humeral Dystrophy (FSHD2), Bosma Arhinia and Microphthalmia Syndrome (BAMS), and the more recently isolated hypogonadotrophic hypogonadism (IHH) combined pituitary hormone deficiency (CPHD) and septo-optic dysplasia (SOD). However, it remains unclear why certain mutations lead to a specific muscle defect in FSHD while other are associated with severe congenital anomalies. To gain further insights into the specificity of SMCHD1 variants and identify pathways associated with the BAMS phenotype and related neural crest defects, we derived induced pluripotent stem cells from patients carrying a mutation in this gene. We differentiated these cells in neural crest stem cells and analyzed their transcriptome by RNA-Seq. Besides classical differential expression analyses, we analyzed our data using MOGAMUN, an algorithm allowing the extraction of active modules by integrating differential expression data with biological networks. We found that in BAMS neural crest cells, all subnetworks that are associated with differentially expressed genes converge toward a predominant role for AKT signaling in the control of the cell proliferation–migration balance. Our findings provide further insights into the distinct mechanism by which defects in neural crest migration might contribute to the craniofacial anomalies in BAMS