Pierre Klein

Immunofluorescence analysis remains a cornerstone technique for evaluating the fate of transplanted human cells in xenograft models, as it provides essential spatial and phenotypic information unavailable through molecular quantification alone. This chapter outlines a standardized procedure for the preparation of human myogenic cells prior to intramuscular injection and then describes a comprehensive immunofluorescence-based method to identify, localize, and quantify human myogenic cells engrafted into regenerating skeletal muscle of immunodeficient mice. Using human-specific antibodies combined with extracellular matrix markers, this approach enables discrimination and classification of engrafted human cells as myonuclei, interstitial cells, or incorporated into the satellite cell niche. Quantitative strategies are presented to estimate total human cell numbers and assess longitudinal and transverse dispersion within host muscle tissue.

PABPN1 est une protéine de liaison à l'ARN nucléaire et ubiquitaire impliquée dans de nombreux mécanismes de régulation post-transcriptionnelle des ARN. Une expansion anormale de triplets GCN dans le gène PABPN1 conduit à une dystrophie musculaire appelée dystrophie musculaire oculopharyngée (OPMD). A l'heure actuelle les mécanismes moléculaires conduisant d'une expansion de quelques triplets dans le gène d'une protéine ubiquitaire vers cette pathologie où seulement quelques muscles sont touchés ne sont pas connus. La caractéristique phénotypique majeure de la maladie est la présence d'agrégats intranucléaires dans les noyaux des muscles atteints et non atteints. Cependant le rôle exact de ces agrégats et leur implication dans la pathologie reste encore incertain. A l'heure actuelle il n'y a pas de traitements curatifs pour l'OPMD. Dans ce contexte, ce projet de thèse a porté sur 1) l'étude des mécanismes moléculaires dérégulés dans la pathologie, 2) l'étude de la contribution des agrégats nucléaires et 3) le développement d'un stratégie de thérapie génique. Au cours de ce travail il a été mis en évidence des défauts mitochondriaux et les mécanismes moléculaires sous-jacents ont été élucidés. L'étude de l'implication des agrégats dans la maladie a révélé la présence de défauts d'épissage et en particulier dans un ARN muscle spécifique codant la protéine TNNT3. Le projet de thérapie génique dit de remplacement consiste à éteindre PABPN1 par interférence à ARN et amener un ADNc (PABopt) qui code la forme saine et qui est non ciblée par les outils interférence grâce à la redondance du code génétique. Les résultats obtenus ont été très positifs à la fois in vitro et in vivo

Background: Myogenic differentiation of muscle stem cells required for skeletal muscle growth and regeneration relies on the precise spatiotemporal coordination of multiple gene-expression programs. Over the past decades, considerable progress has been made in defining the transcriptional networks that govern myogenesis. In particular, myogenic transcription factors integrate extrinsic and intrinsic cues to drive skeletal muscle development and regeneration. In contrast, post-transcriptional layers of regulation have remained comparatively underexplored. Among these, the epitranscriptome – comprising more than 170 chemical RNA modifications – has recently emerged as a major regulatory axis in cellular differentiation. N⁶-methyladenosine (m⁶A), the most abundant internal modification of mRNA, is now recognized as a key regulator of stem-cell fate decisions across multiple biological systems. By dynamically modulating RNA stability, translation, and other processing steps, m⁶A enables precise fine-tuning of gene-expression programs in response to physiological cues. These effects are mediated through m⁶A-binding RNA-binding proteins (RBPs), also referred to as “readers”, which selectively recognize methylated transcripts and translate the modification into functional cellular outcomes. Despite its central role in other tissue contexts, the contribution of m⁶A to skeletal muscle physiology – and particularly to human muscle stem cell biology – remains poorly characterized. This gap represents a critical and largely unexplored frontier in muscle biology. Methods: To define the epitranscriptome landscape during the myogenic differentiation of human skeletal muscle cells, we performed GLORI on proliferating and differentiated human primary myoblasts. This recently developed approach enables detection of m⁶A at single-nucleotide resolution together with quantitative measurement of methylation stoichiometry at individual sites. m⁶A-modified positions were mapped across the transcriptome in both cellular states and integrated with matched RNA-seq datasets data to contextualise m⁶A features relative to transcript abundance. To place these findings in a broader context, we compared our human m⁶A maps with previously published mouse muscle cell datasets and overlaid them with available CLIP-seq datasets of m⁶A RNA-binding proteins. Results: We identified tens of thousands of high-confidence m⁶A sites in proliferating and differentiated human myogenic cells. While many sites were shared between states, myogenic differentiation was accompanied by epitranscriptomic remodeling, affecting both the distribution and stoichiometry of m⁶A marks. Transcripts exhibiting differential m⁶A regulation were enriched for pathways related to myogenic development, cytoskeletal remodeling, signalling, nucleic-acid–associated processes and energy metabolism. Joint analysis of m⁶A site number, methylation stoichiometry, and expression levels indicated the presence of distinct m⁶A regulatory frameworks at the transcript level. Genes associated with myogenesis and regulatory pathways were distributed across these categories, consistent with the coexistence of multiple regulatory programs during myogenic differentiation. In addition, cross-species comparisons revealed the presence of human-specific methylation patterns. Integration with published m⁶A RNA-binding protein datasets uncovered distinct subsets of m⁶A sites with varying binding profiles, suggesting state-dependent interpretation of m⁶A marks. Together, these data constitute, to our knowledge, the first single-nucleotide–resolution atlas of m⁶A methylation in human skeletal muscle, revealing a previously uncharacterized epitranscriptomic landscape and providing a foundational resource for studies of human myogenesis.

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate gene expression programs shaping neuronal morphology are poorly understood. To address this, we studied how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the human neuronal differentiation. We perform a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we show that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate expression of the gene programs shaping neuronal morphology are poorly understood. To address this, we analysed how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the differentiation of human neurons. We performed a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we showed that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11–18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence’ and `replace’ vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence’ and `replace’ cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

N⁶-methyladenosine (m⁶A) is the most prevalent modified nucleotide in mRNA^1,2, with around 25% of mRNAs containing at least one m⁶A. Methylation of mRNA to form m⁶A is required for diverse cellular and physiological processes³. Although the presence of m⁶A in an mRNA can affect its fate in different ways, it is unclear how m⁶A directs this process and why the effects of m⁶A can vary in different cellular contexts. Here we show that the cytosolic m⁶A-binding proteins—YTHDF1, YTHDF2 and YTHDF3—undergo liquid–liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m⁶A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA–YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m⁶A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m⁶A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m⁶A-modified mRNAs are governed by liquid–liquid phase separation principles.

Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell- and gene- based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space. These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein.