Disease exacerbation in MYOrganoids derived from Duchenne Muscular Dystrophy iPSC reveals limitations of microdystrophin therapeutic efficacy

Disease exacerbation in MYOrganoids derived from Duchenne Muscular Dystrophy iPSC reveals limitations of microdystrophin therapeutic efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disease exacerbation in MYOrganoids derived from Duchenne Muscular Dystrophy iPSC reveals limitations of microdystrophin therapeutic efficacy Sonia Albini, Laura Palmieri, Louna Pili, Abbass Jaber, Ai Vu Hong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4270736/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Current gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin (µDys) have shown limited efficacy in patients, contrasting with the favorable outcomes observed in animal models. This discrepancy is partly due to the lack of models that replicate key pathogenic features associated with the severity of the human disease, such as fibrosis and muscle dysfunction. To tackle the translational gap, we develop a human disease model that recapitulates these critical hallmarks of DMD for a more predictive therapeutic investigation. Using a muscle engineering approach, we generate MYOrganoids from iPSC-derived muscle cells co-cultured with fibroblasts that enable functional maturation for muscle force analysis upon contractions. Incorporation of DMD fibroblasts within DMD iPSC-derived muscle cells allows phenotypic exacerbation by unraveling of fibrotic signature and fatiguability through cell-contact-dependent communication. Although µDys gene transfer partially restores muscle resistance, it fails to fully restore membrane stability and reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our human DMD system, an unparalleled aspect in existing DMD models, and provide the opportunity to explore the underlying mechanisms of dysregulated cellular communication to identify anti-fibrotic strategies empowering gene therapy efficacy. Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Biotechnology/Gene therapy/Targeted gene repair iPSC DMD fibroblasts fibrotic MYOrganoids AAV microdystrophin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Duchenne muscular dystrophy (DMD; ONIM: #310200) is an X-linked disorder that affects one in every 5000 male births 1 with no resolutive cure up to date. It is characterized by progressive muscle wasting affecting skeletal muscles primarily and cardiac and respiratory muscles later, thereby causing premature death 2 . DMD is caused by genetic mutations in the DMD gene, leading to the absence of Dystrophin, an essential protein that provides physical support to myofibers by linking them to the extracellular matrix through the Dystrophin Glycoprotein Complex (DGC) 3 – 6 . The lack of Dystrophin results in a series of muscle membrane breakdowns and repairs, leading to subsequent secondary issues like chronic inflammation and fibrosis 7 – 10 . Fibrosis is an excessive deposition of extracellular matrix components like fibronectin and collagen, triggered by overactivation of Transforming Growth Factor beta (TGF-β) and leading to loss of muscle functionality 12 – 13 . Besides being a critical driver of DMD progression, fibrosis also hampers gene therapy efficacy and is therefore paramount to counteract this process that is well-established in patients. Gene therapy using adeno-associated virus (AAV) is currently the most promising treatment for Duchenne muscular dystrophy. Ongoing clinical trials use AAV to deliver short forms of Dystrophin, known as microdystrophin (µDys), which encodes a truncated but functional protein 13 – 19 . However, while the therapeutic effects were unequivocally achieved in DMD animal models 20 , the results from clinical trials revealed only partial therapeutic efficacy in terms of gain of muscle function and rarely addressed whether fibrotic activity and signaling were reduced by gene transfer 21 , 22 . These observations confirm the limited translatability of results obtained in animal models to human patients. It appears therefore crucial to develop time and cost-effective high throughput models, mimicking the severity of human DMD pathology, suitable for research investigation and therapeutic screening. In this context, in vitro modeling based on human cells is a valuable option. In particular, the induced pluripotent stem cells (iPSC) technology offers the opportunity to derive an unlimited number of specialized cells from patients for disease modeling and drug screening 23 , 24 . Among the in vitro cellular models, organoid-like structures are becoming invaluable for disease modeling as the use of 3D cultures and biomaterials allows the reconstitution of tissue architecture and microenvironment that are instrumental for pathophysiological evaluations 25 , 26 . Tissue engineering applications for AAV gene therapy have been exploited mostly in the context of retinopathies 24 , 27 – 29 while only limitedly explored for muscular disorders. Hence, having human DMD models is of utmost importance to advance gene therapy and provide a platform for predictive screening. Although several in vitro 3D systems are accessible for modeling Duchenne muscular dystrophy 30 – 33 , their throughput use is limited by the long duration, variability, related to the complexity of cellular composition achieved, and lack of disease-specific readouts for muscle function. Here, we report on the generation of iPSC-derived muscle organoid structure, named hereafter MYOrganoids. We employ and adapt an engineered muscle platform to generate MYOrganoids using a previously reported method for direct iPSC conversion into 2D skeletal muscle cells 34 – 36 . As a strategy to increase the structural and functional maturation required for pathophysiological studies, we use fibroblasts as they are a major source of connective tissue which is a key regulator of differentiation and muscle structure. Moreover, fibroblasts act as a source of microenvironment cues exerted by their secretory activity and they are therefore regulators of the muscle niche that undergoes pathological remodeling during disease 37 , 38 .Here we show that fibroblast inclusion enhances the structural and functional maturation of the muscle cells. In a DMD context, fibroblasts allow exacerbation of phenotypic traits by direct interaction with muscle cells and reveal key hallmarks of DMD such as fibrosis and muscle weakness over repeated contractions. Our study also evaluates for the first time the therapeutic efficacy of AAV-mediated µDys gene transfer, in engineering muscle tissues, as proof of concept of their suitability for studying disease mechanisms and evaluating potential therapeutics. By using different doses of µDys in DMD MYOrganoids, we observed a dose-dependent response in restoring muscle function while only a partial effect at the level of membrane stability and fibrotic signature in DMD muscles. Our findings indicate that patient-derived MYOrganoids, whose pathogenic traits are exacerbated, are suitable for studying the fibrotic process orchestrated by either muscle or fibroblast population and its interplay with gene transfer approaches. Our system has therefore the potential to identify molecular mechanisms driving the dystrophic process and accelerate the identification of effective therapeutics for DMD. Results Generation of structurally organized 3D human MYOrganoids by direct conversion of iPSC and inclusion of fibroblasts MYOrganoids were generated from human iPSC committed to differentiating into the myogenic lineage by inducible expression of MyoD and BAF60C 34 (referred to as iPSC BM .) that can directly generate myotubes. MYOrganoids were prepared starting from iPSC BM after one day from the induction of myogenic genes. The casting procedure was performed through adaptation of an engineered muscle system 33 , 39 which results in the growth of the tissue in a ring format supported by two flexible silicon stretchers. The 3D cultures were kept for 2 days in growth medium, afterwards medium was replaced for differentiation for another 12 days (Fig. 1 A). The differentiation protocol was optimized from the conditions previously reported 35 , 36 , 40 using myogenic commercial media that ensured the highest expression of myogenic markers and myogenic differentiation in monolayer conditions ( Figure S1 A-B ). Since cellular heterogenicity, especially of mesenchymal origin, is important for muscle formation 33 , 37 , 38 , 41 , we included human fibroblasts during the casting procedure, to assess whether this would affect muscle organization. For this aim, casting was performed using iPSC BM cells in the presence or absence of human fibroblasts. Achieving alignment and differentiation simultaneously necessitates a delicate balance between fibroblasts and muscle cells (as noted by N. Rao et al., 2013). To replicate physiological conditions accurately, we incorporated a fibroblast concentration that mirrors the stromal population detectable through single cell and single nuclei-RNA seq analysis of muscles 42 – 45 . We found that including 10% fibroblasts, accelerated the condensation and growth over time of the muscle rings into a compact structure 0.8 mm long and 1 mm thick at day 14 (Fig. 1 B ). By performing immunofluorescence in whole-mount tissues for sarcomeric α-actinin (SAA), we could detect an enrichment of SAA-positive myotubes throughout the ring-shaped micro-tissue (Fig. 1 C). We then aimed to assess the impact of fibroblast inclusion on muscle structure. Since the organization of muscle cells within the ECM plays a key role in fusion and maturation 46 , both alignment of myotube and circularity, a consequence of their parallelism, were evaluated. Staining for myosin heavy chain (MyHC, myotube marker) and vimentin (fibroblasts marker) on longitudinal sections showed fibroblast recruitment near muscle fibers (Fig. 1 D). Myotubes alignment was determined by measuring the angles in between myotubes 47 and showed a significant decrease towards 0 degrees upon fibroblast inclusion, which indicates parallelism while, in the MYOrganoids without fibroblasts, we detected a disordered pattern of muscular cells (Fig. 1 E). Additionally, the circularity of myofibers was measured from transversal sections stained for the membrane marker wheat germ agglutinin (WGA), using the ratio between X and Y Feret diameters (Fig. 1 D). MYOrganoids including fibroblasts had an improved circularity (ratio closer to 1) when compared to control (Fig. 1 E). Improved myotube circularity and alignment as shown, indicate that fibroblast incorporation during the casting procedure guides skeletal cell orientation providing structural support for MYOrganoids, a prerequisite for maturation. Increased structural and functional maturation of fibroblast-including MYOrganoids Since muscle maturation is strictly dependent on the internal myofiber organization, we evaluated the differentiation of our 3D MYOrganoids by looking at the sarcomere structure. Transversal and longitudinal sections were used to monitor dystrophin (DYS) expression at the sarcolemma ( Fig. 2 A) and sarcomeric α-actinin (SAA) localization for assessment of the striation pattern typical of mature myotubes (Fig. 2 B). Dystrophin was properly localized to the muscle membrane of myotubes from MYOrganoids including fibroblasts and was significantly more expressed than MYOrganoids without fibroblasts (Fig. 2 D). Remarkably, the maturation index, reported as a percentage of the number of nuclei included in striated myotubes, was significantly superior in MYOrganoids including fibroblasts as compared to MYOrganoids without fibroblasts which appear very disorganized with a rare appearance of striations (Fig. 2 E). Increased maturation of myotubes within MYOrganoids including fibroblast was also supported by analysis of the fusion index, indicating a significantly higher percentage of multinucleated myotubes (> 3 nuclei) and a lower percentage of mononucleated ones (Fig. 2 F). This evidence highlights the positive role of fibroblasts in the maturation process through fusion and multinucleation. The proper sarcomeric organization was also confirmed by electron microscopy where we could detect longer, properly formed Z patterning and the presence of I and A bands along with an overall increase of sarcomeric density and alignment (Fig. 2 C). Consistently, MYOrganoids containing fibroblasts showed wider Z-line (Fig. 2 H), index of higher maturation of sarcomeres 48 . We further performed gene expression analysis for terminal differentiation markers such as muscle creatine kinase ( MCK ), myosin heavy chain ( MYH ) isoforms, such as MYH2 , representative of fast adult fiber type, and MYH7 , as a slow fibers marker (Fig. 2 G). Higher expression of all genes in MYOrganoids containing fibroblasts confirms the acquisition of a more mature state, compared to MYOrganoids without fibroblasts. We then assessed whether our MYOrganoids were functional by evaluating their physiological response to contraction stimulations, using a muscle organ bath system based on electrical pacing 49 . To evaluate muscle force, MYOrganoids were transferred to the muscle strip chamber and stretched until the optimal length (Lo) for functional analysis (Fig. 2 I). Isometric force analysis revealed significantly higher tetanic force in MYOrganoids containing fibroblasts compared to the control (Fig. 2 J). Values were then normalized for the cross-sectional area (CSA) using the weight and optimal length of contraction established for each MYOrganoid 50 , 51 and expressed as specific tetanic force (mN/mm 2 ) (Fig. 2 K). In particular, the highest force with fibroblasts had peak values ranging from 0.3 to 0.5 mN versus 0.1 to 0.2 mN in MYOrganoids without fibroblasts after normalization (Fig. 2 K). These data demonstrated that MYOrganoids plus fibroblasts have an improved structural organization and functional maturation that enables force contraction studies by electrical pacing. Inclusion of fibroblasts in DMD iPSC-derived MYOrganoids leads to increased muscle fatigue and pro-fibrotic signature The improved muscle organization and functional maturation shown by MYOrganoids including fibroblasts, prompted us to exploit fibroblast features in disease modeling for DMD, where their role in disease progression is well known 11 , 12 . We incorporated DMD fibroblasts to recapitulate the pathogenic microenvironment arising from their profibrotic activity exerted by tissue remodeling and matrix deposition 12 , 52 . For that purpose, we used three DMD iPSC with different DMD mutations, a deletion of exon 45 (DMDdEx45), a deletion of exons 8–43 (DMDdEx8-43) and a deletion of exons 8–9 (DMDdEx8-9) with their isogenic control, the DMD dEx6-9 iPSC corrected to restore dystrophin expression 53 (hereafter called IsoCTR). We additionally used two control iPSC lines derived from healthy patients (CTR1, CTR2). Control and DMD MYOrganoids were generated from control and DMD iPSC with healthy or DMD human immortalized fibroblasts for functional and transcriptomic analysis (Fig. 3 A). The myogenic differentiation ability of both CTR and DMD iPSC lines was first evaluated in 2D cultures. All cell lines showed comparable myogenic potential, as expected using a direct myogenic conversion protocol bypassing any defective developmental steps ( Figure S2A ). Histological characterization in MYOrganoids showed the absence of dystrophin protein in DMD MYOrganoids and efficient myogenic differentiation and maturation in CTR and DMD MYOrganoids, as shown by the striated pattern visualized by SAA-stained sections, confirming the positive role of fibroblasts in enhancing maturation also in a DMD context (Fig. 3 B). To assess whether DMD MYOrganoids display hallmarks of DMD pathophysiology, we evaluated muscle function, which represents one of the most difficult challenges in establishing therapeutic readouts with in vitro systems. To identify reliable force parameters reflecting the defective DMD muscle performance, MYOrganoids were subjected to isometric contractions to measure tetanic force, and to eccentric contractions, which play a critical role in the disease progression of DMD 54 by triggering the membrane degeneration/regeneration cycles 55 to assess muscle strength and fatiguability. No significant changes were observed in the tetanic isometric force between CTR and DMD MYOrganoids ( Figure S2B ), as expected for muscles never been challenged by contractions and with the same myogenic potential, while DMD MYOrganoids showed higher drop force in the eccentric contraction repetitions exclusively when including DMD fibroblasts starting from the 4th eccentric repetition (Fig. 3 C). On the other side, CTR and DMD MYOrganoids not including fibroblasts, show significant differences in drop force just at the tenth repetition (Fig. 3 C). To accurately quantify muscle fatigue, we calculated the fatigue index as the drop of force between the isometric contractions performed before and after the 10 repetitions of eccentric exercise. The analysis showed a significantly higher fatigue index in DMD MYOrganoids as compared to CTR MYOrganoids and remarkably, this phenomenon was highly accentuated by the presence of fibroblasts. (Fig. 3 D ) . To check whether the impact on disease exacerbation and increase of difference in fatiguability between CTR and DMD MYOtissue was affected by the different genetic backgrounds of the fibroblast source, we employed three additional fibroblast cell lines from either healthy and DMD individuals to generate CTR and DMD MYOrganoids. Muscle force analysis confirmed that the fatigue index in DMD MYOrganoids was significantly higher than CTR MYOrganoids, regardless of the genetic source of either control or DMD fibroblasts. This analysis also confirmed the display of higher and significant muscle fatiguability in fibroblasts-including MYOrganoids ( Figure S2C ). Collectively, these data indicate that DMD MYOrganoids including DMD fibroblasts, through their pro-fibrotic activity, display exacerbated loss of muscle resistance and increase in fatiguability (Fig. 3 A-D ) . The finding also demonstrates that eccentric-based drop force evaluation is a meaningful and reliable therapeutic readout, as significant differences were detected between CTR and DMD MYOrganoids including fibroblasts. We then performed transcriptomic analysis in the isogenic iPSC lines (IsoCTR and DMDdEx8-9) to better characterize the pathogenic hallmarks introduced by fibroblast incorporation within the organoids. Analysis of significant (p.adj 1) revealed 4610 differentially expressed genes (DEGs) between DMDdEx8-9 and IsoCTR MYOrganoids, of which 2181 upregulated and 2429 downregulated ( Figure S3A-B ). Of those, we identified genes involved in decreased muscular contraction (e.g. muscle system process, muscle contraction, sarcoplasmic reticulum calcium ion transport and actin-myosin filament sliding) as well as reduced energetic molecular process (e.g. oxidative phosphorylation, mitochondrial respiratory chain complex assembly, ATP synthesis coupled electron transport), as depicted by gene ontology biological process (GOBP) enrichment analysis (Fig. 3 E, table S1 ). Remarkably, between the upregulated pathways in DMD MYOrganoids, we identified higher collagen fibril organization with an enrichment score of + 0.6 (Fig. 3 E). This GO analysis was also confirmed by Kyoto encyclopedia of genes and genome (KEGG) enrichment analysis, which showed a downregulation of oxidative phosphorylation and an upregulation of ECM receptor interaction, such as Integrin alpha and beta ( Figure S3C ). Interestingly, the analysis of genes involved in the ECM remodeling revealed an upregulation of genes coding for collagens, laminins, metalloproteases and TGF-β related genes such as TGF-B receptor (TGFBR1) and the ncRNA inhibitor of TGF-β (TGFB2-AS1) in DMD MYOrganoids compared to isogenic control (Fig. 3 F). Additionally, we found increased levels of latent TGF-β binding proteins (LTBP 2 and 4) in dystrophic MYOrganoids, indicating that TGF-β is activated in the DMD context and contributes to ECM remodeling ( Table S1 ). The increased profibrotic signature in DMD iPSC-derived MYOrganoids was then confirmed in all CTR and DMD iPSC-derived MYOrganoids, by evaluation of the expression of two key fibrotic markers, Fibronectin-1 ( FN1 ) and Collagen-1 ( COL1A1 ). Importantly, the differences in the expression of fibrotic markers between CTR and DMD MYOrganoids were significant only when including fibroblasts (Fig. 3 G). We further examined, at histological level, the presence of activated TGF-β signaling in DMD organoids by looking at phosphorylated SMAD3 (pSMAD3), the transcriptional effector of the canonical TGF-β pathway. Notably, DMDdEx8-9-derived MYOrganoids showed increased pSMAD3 positive nuclei both in myotubes (MyHC positive staining) and fibroblasts (MyHC negative cells) (Fig. 3 H). This observation indicates a crosstalk between fibroblasts and myofibers and supports the potential of using fibroblasts in tissue engineering as a source of ECM and pro-fibrotic cues under pathological conditions. Juxtracrine role of fibroblasts in the modeling of DMD phenotype We then sought to gain insights into the contribution of fibroblasts in the recapitulation of key pathogenic hallmarks of DMD. Given that fibroblasts are known to primarily act through their secreted molecules 56 – 58 we analyzed the secretome of both control (CTR) and DMD MYOrganoids (MyoT + Fibro) and compared it to that of single-cell 3D cultures (Fibro-only and MyoT-only) (Fig. 4 A). Analysis of culture media on day 14 showed elevated levels of secreted TGF-β and Collagen 4 (COL4) in DMD MYOrganoids cocultured with fibroblasts compared to control conditions. No significant differences were observed between control and DMD in Fibro-only and MyoT-only 3D cultures (Fig. 4 B). These data indicate that DMD MYOrganoids in coculture with fibroblast exhibit an increased release of fibrotic factors. To determine whether fibroblasts amplify the fibrotic phenotype through their secretory activity, we collected conditioned media (CM) from control (CTR) and DMD Fibro-only 3D cultures on day 7 of in vitro growth. We then applied this CM to treat CTR and DMD MyoT-only tissues for additional 7 days, assessing the secretion of fibrotic factors, gene expression, and muscle force (Fig. 4 C). ELISA assay on secreted TGF-β and fibronectin 1 (FN1) revealed no difference in MyoT-only 3D cultures treated with the CM from either CTR or DMD Fibro-only tissues (Fig. 4 D). Congruently, gene expression levels of TGF-β and Col1A1 were similar in CTR and DMD MyoT-only cultures, regardless of the treatment with the CM (Fig. 4 E), while those were higher just in DMD cocultures (MyoT + Fibro) compared to control conditions ( Figure S4A ). The lack of effect of the CM indicates that the profibrotic increase seen in DMD MYOrganoids in coculture with fibroblasts occurs by a juxtracrine effect, as it comes from contacts between the two cell types. We further evaluated the requirement of fibroblasts-myotubes coculture in disease exacerbation by assessing muscle force analysis following CM treatment, which confirmed no difference in fatiguability in myotube-only 3D cultures treated with control or DMD Fibro-derived CM, unlike higher fatigue index in DMD organoids containing both fibroblasts and myotubes in coculture (Fig. 4 F). Interestingly, MYOrganoids including fibroblasts displayed a higher tetanic force compared to myotubes-only cultures treated or not with fibroblasts CM, also supporting their cell contact-dependent role for enhanced functional maturation ( Figure S4B ). Collectively, analysis of secreted protein and force under CM experiments showed that the dystrophic fatiguability and fibrosis traits emerged only when fibroblasts and muscle cells were cocultured, indicating that contact-dependent cellular communication between muscle and fibroblasts is required to reveal the DMD traits of profibrotic secretion and fatiguability. AAV-microdystrophin gene transfer rescues muscle resistance while partially restoring Dystrophin Glycoprotein Complex components in DMD MYOrganoids As proof of concept that MYOrganoids were suitable as a screening platform for gene therapy, we used AAV-mediated delivery of microdystrophin (µDys) and assessed its therapeutic efficacy in the DMD context. We used AAV9 capsid for this aim, as this viral serotype was used in recent clinical trials and showed a high transduction rate in patients’ myofibers 59 , 60 . We infected the organoids with a codon-optimized µDys gene (dR4-23, same protein product used in clinical trials 61 ) under the control of the muscle-specific spc512 promoter 16 for gene transfer using AAV9 (AAV9-µDys) in all DMD MYOrganoids iPSC (dEx45, dEx8-43 and dEx8-9 with isogenic control, IsoCTR) and assessed gene transfer efficiency, muscle force analysis and membrane stability assessment (Fig. 5 A). We first optimized the infection conditions using the reporter AAV9-CMV-GFP in CTR MYOrganoids including fibroblasts. Infection was performed on day 7 of the differentiation protocol, diluting AAV particles directly in the medium, and maintained for an additional 7 days ( Figures S5A-C ). Optimal low and high AAV9-µDys doses were established based on previous dosing studies to have intermediate transduction levels at low doses (1E + 09 vg/MYOrganoid), and high transduction levels at high doses (5E + 10 vg/MYOrganoid) ( Figure S5D ). Gene transfer efficiency was evaluated by quantification of viral copy number (VCN) on genomic DNA and by the expression level of the transgene and the encoded protein, showing a clear dose-dependent entry and expression of µDys in DMD MYOrganoids (Fig. 5 B, S6A-B). As expected by using a muscle-specific promoter, only the muscle cells (MyoT) but not the fibroblasts expressed the transgene after infection of AAV9-uDys in the single populations ( Figure S6C ). We then tested whether µDys affected the contractility and fatigue resistance of the infected organoids. Isometric tetanic force analysis did not reveal any significant changes in DMD MYOrganoids following µDys delivery compared to not-infected conditions ( Figure S6D ), as expected by first-time contracting muscle tissues. We then challenged the muscles with repeated eccentric repetitions, and we observed a dose-dependent attenuation of the drop force in DMD MYOrganoids after µDys infection (Fig. 5 C). Consistently, the fatigue index was greatly reduced in a dose-dependent manner in all DMD MYOrganoids treated with a high dose of µDys, although reaching significance only for DMDdEx45 and DMDdEx8-9 MYOrganoids (Fig. 5 D). To confirm that the observed effects were specifically due to µDys expression rather than AAV infection alone, we infected DMD MYOrganoids with a control vector containing a dystrophin fragment lacking a promoter (hereafter called empty vector), which infection did not result in protein expression ( Figure S6E-G ). Particularly, the MYOrganoids infected with the empty vector did not show any differences in the fatigue index analysis, at both low and high dose, while we observed a decreased fatigue index in DMD MYOrganoids infected with an AAV containing µDys ( Figure S6H ). Overall, the muscle force data confirmed that ectopic µDys expression rescues muscle endurance and fatiguability. Because dystrophin exerts its biomechanical support by holding the Dystrophin Glycoprotein Complex (DGC) at the membrane, we monitored key components of this complex, such as the transmembrane β-Dystroglycan (β-DG), directly binding Dystrophin, and the extra-cellular α-Dystroglycan (α-DG), whose proper expression and localization are impaired in the absence of dystrophin 62 . To this aim, DMD MYOrganoids infected with low and high dose of µDys, were subjected to histological analysis. Quantification of µDys showed around 25–30% of dystrophin-positive myotubes at low dose compared to 85% in the high dose condition in DMD dEx8-9 (Fig. 5 E-F) and dEx45 and 65% in the DMD dEx8-43 MYOrganoids ( Figure S7A-B ). Immunostaining on transversal MYOrganoids showed a dose-dependent yet not complete restoration of β-DG in all DMD iPSC-derived MYOrganoids. Interestingly, even high doses ensuring nearly total Dystrophin transduction in all the DMD MYOrganoids did not significantly restore α-DG (around 10% in DMDdEx8-9 and DMDdEx8-43 and around 30% in DMDdEx45) suggesting a potential therapeutic limitation of µDys (Fig. 5 E-F). Importantly, these data demonstrate the limited restoration of Dystrophin-associated components to the sarcolemma after successful µDys gene transfer. Optimal microdystrophin gene transfer partially corrects the transcriptomic profile of DMD MYOrganoids and does not rescue the profibrotic signature To examine the effect of microdystrophin (µDys) on the signaling pathways and molecular processes characteristics of dystrophic pathology, we performed bulk RNA-seq analysis comparing the corrected DMD dEx8-9 (IsoCTR) and DMDdEx8-9 MYOrganoids untreated and treated with a high dose of AAV9-µDys (n = 4). Principal component analysis (PCA) of the top 10000 genes with the highest variance showed a distinct transcriptomic profile of DMD organoids treated with µDys (dEx8-9 + µDys high) and untreated (dEx8-9) compared to isogenic controls (IsoCTR) ( Figure S8A ). To evaluate the effect of µDys in restoring key pathways involved in DMD, we firstly compared the transcriptomic profile of µDys-treated DMD organoids to untreated DMD organoids (dEx8-9 µDys vs dEx8-9), and then to the isogenic control (dEx8-9 µDys vs IsoCTR). Particularly, µDys-treated DMD organoids showed a total of 327 DEGs, of which 154 upregulated and 173 downregulated, compared to untreated organoids (Fig. 6 A, Table S2 ). Of those 327 DEGs, 52% (170/327) are in common with the DEGs that were found dysregulated in DMD organoids when compared to the isogenic control ( Figure S8B ). Therefore, we performed the gene ontology enrichment analysis of the common category to see the effect of µDys on the DMD-dysregulated pathways. This analysis revealed increased expression of genes involved in the microtubule organization (GOBP: Microtubule organizing center organization NES = + 2.31), cytoskeleton organization (GOBP: Protein localization to cytoskeleton NES = + 1.99), and a decreased expression of genes involved in the inflammation (GOBP: Regulation of inflammatory response, NES = -1.45) (Fig. 6 B). We then analyzed the DEGs between the treated organoids and the isogenic control to check the transcriptomic signature not restored by µDys (dEx8-9-µDys vs IsoCTR). Particularly, µDys-treated organoids present 7818 DEGs (4004 upregulated and 3814 downregulated) when compared to the corrected isogenic organoids (Fig. 6 C). Gene ontology analysis of the DEGs between µDys-treated DMD organoids and IsoCTR organoids revealed the pathways that are not restored by the gene transfer. Particularly, treated organoids showed persistent downregulation of genes involved in calcium transport (GOBP: Sarcoplasmic reticulum calcium ion transport, NES = -2.15), oxidative phosphorylation (GOBP: Oxidative phosphorylation, NES = -2.29) and upregulation of TGFβ signaling (Hallmark TGFβ signaling, NES = + 1.57), when compared to isogenic controls (Fig. 6 D, Table S3 ). Consistently, a clustered heatmap of all 1716 most significant differentially expressed genes (DEGs) further highlighted the transcriptomic differences between control and DMD organoids (Fig. 6 E). Analysis of Z-score of fibrotic genes among the three conditions (IsoCTR, DMDdEx8-9 and DMDdEx8-9 treated with µDys) revealed a persistent but variegated profibrotic signature. In particular, we revealed a slight decrease in the expression of ADAMs (i.e. ADAM17, ADAMTS7, ADAMTS16), collagen genes (e.g. COL11A1, COL4A6) and the TGF-β receptor 1 gene (TGFBR1) in µDys-treated DMD organoids, when compared to the untreated condition, although still significantly different from isogenic control (Fig. 6 E, right panel). Additionally, other fibrotic genes, like FN1 and COL1A 1, Integrins or cytokines (e.g.IL1A), showed no expression changes compared to untreated control (Fig. 6 E, right panel ). Further validation of the expression of COL1A1 , FN1 , not decreased by the µDys treatment, and of TGF-β , partially downregulated, confirmed the persistent expression of those genes in DMD-treated MYOrganoids (Fig. 6 F). To understand the mechanisms behind the unresolved fibrotic process, we evaluated the reduction in TGF-β pathway activation within both cell subpopulations in DMD MYOrganoids following µDys treatment. Histological staining revealed a significant decrease in pSMAD3-positive nuclei within the skeletal muscle subpopulation of µDys-treated organoids, whereas no difference was observed in fibroblasts after µDys treatment (Fig. 6 G). This result suggests a cell-autonomous correction of this signaling within the muscle population receiving µDys, but no paracrine effect on fibroblast activity. The persistence of activated TGF-β signaling in the fibroblast subpopulation after µDys gene transfer (Figs. 3 H and 6 G) can therefore explain the uncorrected profibrotic signature and poses the basis for future investigation aimed at improving or driving beneficial cell-cell communication. Discussion Here, we present a novel human-relevant model that simulates key traits of advanced stages, enabling the interrogation of gene replacement effect on muscle function and fibrosis, and facilitating the identification of new therapeutic avenues. We report the generation of 3D-engineered muscles called MYOrganoids, composed of a homogeneous population of iPSC-derived skeletal muscle cells 35 , 40 and fibroblasts, major players in muscle tissue organization and microenvironment regulation 37 , 38 , 63 . We proved that fibroblast inclusion in the MYOrganoids results in improved structural maturation that allows high functional performance under muscle force evaluation, while also eliciting a fibrotic signature in a DMD context. Importantly, fibroblast incorporation was pivotal in exacerbating DMD phenotype and revealing fatiguability, which is a top hallmark for DMD muscle function, thereby enabling its unprecedented use as therapeutic readouts in vitro. This is important considering that the capacity to evaluate muscle function in a high throughput manner remains limited, further hindering progress in testing or developing effective treatments. As such, while several remarkable studies reported on muscle force in 3D models 32 , 33 , 53 , 64 , 65 , evaluating muscle function still presents challenges due to the complexity of identifying disease-specific force parameters and the variability of in vitro models. Furthermore, our study sheds new light on the mechanism underlying the contribution of fibroblasts to the modeling of DMD phenotype. We showed that direct interaction of DMD fibroblast with muscle cells is essential to reproducing key pathogenic hallmarks, such as a profibrotic signature and increased muscle fatigue in DMD MYOrganoids. Interestingly, fibroblast role in the recapitulation of DMD hallmarks was not exerted by their profibrotic secretory activity per se but required direct interaction with iPSC-derived muscle cells. As such, fibroblasts in a DMD context showed higher secretion of fibrotic factors like TGF-β, fibronectin, and collagen through cell-contact signaling, suggesting that cell communications between the two cell populations, fibroblasts and muscle fibers, is crucial to reveal pathogenic traits. One possible hypothesis is that fibroblasts secretory activity is enhanced by alterations in cell communications with dystrophin-deficient muscle cells whose membrane stability is impaired, thereby causing increased matrix deposition and susceptibility to contraction-induced damage and fatigue. Overall, the exacerbated severity of DMD MYOrganoids enabled the evaluation of the therapeutic potential of µDys, currently employed in clinical trials. Consistently with the partial efficacy in the clinics, µDys 16 , 17 delivery in DMD MYOrganoids did not fully rescue DMD phenotype as it led to incomplete restoration of the components of the dystroglycan complex and no reduction in fibrotic genes transcriptome. Notably, we observed a reduction in the TGF-β pathway activation exclusively on muscle cells, while no effect was observed in fibroblasts. This can explain the persistence of ECM protein production and secretion in µDys treated DMD MYOrganoids, while we observed an effect in the restoration of muscle strength. Our findings align with previous results showing that µDys exhibits limited effectiveness in reducing fibrosis in advanced disease stages 66 and in more severe models and muscles affected by the disease, such as the diaphragm in the Dba2 mdx model 67 . We can speculate that the partial restoration of DAG complex after gene transfer causes mechanical alterations of the membrane that are translated as profibrotic signals. In advanced disease stages, the stiffened ECM perpetuates fibrosis through mechanotransduction, activating fibroblasts to deposit ECM proteins and increasing rigidity. This feedback loop hinders microdystrophin's ability to reverse fibrosis. Up to now, no other work revealed the impact of µDys on the fibroblast population of the muscle, revealing the potential of our system in highlighting specific cell-cell communications in response to treatment. Further work is needed to fully understand the role of fibroblasts in the dystrophic process. For example, how the absence of dystrophin in the myotubes is influencing fibroblast activity, and how the reintroduction of µDys is affecting the composition of the ECM, still needs to be elucidated. Overall, pro-fibrotic DMD MYOrganoids provide a valuable tool either for the investigation of mechanisms driving dystrophic process and as a human in vitro counterpart to animal in vivo preclinical studies, with the potential to unravel therapeutic limitations of current DMD treatments and accelerate the identifications of new treatments. Declarations Acknowledgments We express our gratitude to Rene Hummel (DMT), Guillaume Tanniou, and Nicolas Guerchet for their invaluable technical support in muscle force evaluation. We also extend our appreciation to Jérémie Cosette for the assistance in imaging and to the histology team for their technical expertise. Finally, we would like to thank Frederique Magdinier for generously providing the DMD iPSC dEx45 and Ctr1 iPSC. Funding This study was financially supported by the Institut National de la Sante et de la Recherche Medicale (INSERM) and by the “Association Française contre les Myopathies” (AFM). Author contributions SA and LPa designed the experiments. LPa performed most of the experiments, including iPSC culture, organoid generation, imaging, and muscle force analysis. LPi performed force analysis, gene expression analysis and western blot experiments. AJ analyzed conditioned media biomarkers and performed secretome analysis. LPa, MM contributed to muscle force analysis. AVH analyzed RNA-seq data. RE and GB performed electron microscopy analysis. AB generated and provided the immortalized human fibroblasts. SA and LPa performed data analysis and figure preparation. SA conceived, supervised the project, and wrote the manuscript. All authors discussed the results. SA, LPa, DI and IR reviewed and edited the manuscript. Declaration of interests All other authors declare they have no competing interests. RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sonia Albini ( [email protected] ). Material availability Ctr2, corrDMDdEx8-9, DMDdEx8-9 and DMDdEx8-43 hIPS cell lines used in this study are restricted due to MTAs (Ref: MTA 2022 GENETHON - AIM _DMD, MMTA202109-0107, 2021-0296_MTA_CECS). This study did not generate new unique reagents. Data and code availability The data supporting the findings of this study are available within the article and its supplemental information. The RNA sequencing datasets generated in this study can be found in the NCBI Bioproject database (https://www.ncbi.nlm.nih.gov/bioproject) using the access number PRJNA1044721. Microscopy and functional data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Experimental Models Induced pluripotent stem cells (iPSCs) Human iPSCs used in the study were as follows: Ctr1 (AG08C5) derived from healthy fibroblasts (AG08498, Coriell Institute), Ctr2 (M180, CECS), IPSC DMDdEx45 (Coriell, GM25313); DMDdEx8-43 (M202, CECS) DMDdEx8-9 and corrDMDdEx8-9 (CRISPR-mediated skipping of mutated exons dEx6-9, isogenic control) (provided by Doctor E. Olson). iPSCs were maintained using mTeSRplus medium (Stem Cell Technologies) and passaged using ReleSR (Stem Cell Technologies) on Matrigel coated wells (Corning). iPSC engineering and muscle differentiation were performed adapting a transgene-based method previously described (Caputo et al. 2020; Albini et al. 2013). Quality control was performed regularly with Oct-4 and Nanog expression profile and mycoplasma testing and cells were used within 30 passages. Additionally, antibiotic and antimycotic solution (Merk-Millipore) was regularly added to each cell culture media. All iPSC lines are engineered for transgene-based myotubes differentiation. Briefly, cells were nucleofected (4D Nucleofector, Lonza) with two enhanced versions of piggyBAC (ePB) containing respectively MyoD and BAF60c genes under tetracycline responsive promoter (Caputo L et al 2020). Additionally, iPSCs were regularly selected for the presence of the two ePBs with Puromycin 10 µg/ml and Blasticidin 20 µg/ml (Sigma-Aldrich). Human immortalized fibroblasts Human immortalized fibroblasts from control (AB1191, AIM) and DMD patient (AB1024, AIM) were generated and obtained from Myobank-AFM of Myology Institute from skin biopsies. Control and DMD human immortalized fibroblasts, used in co-culture with hiPSC in 3D MYOrganoids, were maintained in culture in DMEM High Glucose Lglut supplemented with 20% fetal bovine serum (FBS) and passaged using TrypleE (Gibco). Human primary fibroblasts Human primary fibroblasts from control (3 lines: 5-8898, 11-1610, 11-1973) and from DMD patients (3 lines, 5-11618, 5-12608, 5-11613) were obtained from DNA bank of Genethon. Fibroblasts were maintained in DMEM high glucose (Gibco) supplemented with 20 ng/ml recombinant human fibroblast growth factor (h-FGF) (Peprotech) and 20% fetal bovine serum (Gibco). Primary fibroblasts were passaged using TrypleE (Gibco) REAGENT / RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Myosin Heavy Chain (used at 1:10) DSHB Cat# MF20, RRID:AB_2147781 Rabbit polyclonal Vimentin (used at 1:100) Proteintech Cat#10366-1-AP, RRID:AB_2273020 Mouse monoclonal Dystrophin (used at 1:20) Leica Biosystem Cat# NCL-DYSB, RRID:AB_563691 Mouse monoclonal Fibronectin (used at 1:200) Sigma-Aldrich Cat# F7387, RRID:AB_476988 Rabbit monoclonal pSMAD-3 (used at 1:100) Abcam Cat# ab52903, RRID:AB_882596 Mouse monoclonal α-Dystroglycan (used at 1:100) Millipore Cat# 05-298, RRID:AB_309674 Mouse monoclonal β-Dystroglycan (used at 1:100) Leica Biosystems Cat# NCL-b-DG, RRID:AB_442043 Rabbit monoclonal alpha-actinin 2 (used at 1:100) Thermo Fisher Scientific Cat# 701914, RRID:AB_2688290 Mouse monoclonal Oct3/4 (used at 1:200) Santa Cruz Biotechnology Cat# sc-5279, RRID: AB_628051 Rabbit monoclonal to cleaved Caspase-3 (used 1:500) Cell Signaling technology Cat# 9664, RRID: AB_ 2070042 Mouse monoclonal Pax7 (used at 1:100) DSHB Cat# PAX7, RRID:AB_2299243 Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21125, RRID:AB_2535767 Goat anti-Rabbit IgG (Heavy chain), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A27034, RRID:AB_2536097 Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21135, RRID:AB_2535774 Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 Thermo Fisher Scientific Cat# A-21242, RRID:AB_2535811 Chemicals, peptides, and recombinant proteins Doxycycline Sigma-Aldrich Cat# D9891-5G Matrigel (Growth Factor Reduced) Corning Cat# 356231 Collagen 6 mg/ml Sigma-Aldrich Cat# 804622-20ML Stemgent hES Cell Cloning & Recovery Supplement Ozyme Cat# STE01-0014-500 Puromycin dihydrochloride Sigma-Aldrich Cat# P8833-10MG Blasticidin Sigma-Aldrich Cat# SBR00022-1ML NaOH Sigma-Aldrich Cat# 221465 Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 488 Invitrogen Cat# W11261 Hoechst 33342 Invitrogen Cat# H3570 TrypLE Express Enzyme Gibco Cat# 12605010 ReleSR Stem Cell Tech Cat# 100-0484 Fetal bovine serum Gibco Cat# 16000044 Goat Serum Dako Cat# X0907 Critical commercial assays MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A - Immunology Multiplex Assay Merck-Millipore Cat# HCYTA-60K Anti-Mouse Detection Module for Jess, Wes, Peggy Sue or Sally Sue Bio-techne Cat# DM-002 RNeasy Micro kit Qiagen Cat# 74404 QX200™ ddPCR™ EvaGreen Supermix Biorad Cat# 1864034 QX200™ ddPCR™ Supermix for Probes Biorad Cat# 1863026 Deposited data RNA sequencing data NCBI BioProject database PRJNA1044721 Experimental models: Cell lines Ctr1 hIPSC Coriell AG08C5/ AG08498 Ctr2 hIPSC CECS M180 corrDMDdEx8-9 hIPSC Kyrychenko et al N/A DMDdEx45 hIPSC Coriell GM25313 DMDdEx8-9 hIPSC Kyrychenko et al N/A DMDdEx8-43 hIPSC CECS M202 Ctr skin fibroblasts immortalized AIM AB1191 DMD skin fibroblasts immortalized AIM AB1024 Primary skin fibroblasts control 1 DNA Bank Genethon 5-8898 Primary skin fibroblasts control 2 DNA Bank Genethon 11-1610 Primary skin fibroblasts control 3 DNA Bank Genethon 11-1973 Primary skin fibroblasts DMD 1 DNA Bank Genethon 5-11618 Primary skin fibroblasts DMD 2 DNA Bank Genethon 5-12608 Primary skin fibroblasts DMD 3 DNA Bank Genethon 5-11613 Oligonucleotides Primers for ddPCR (see table S5) This paper N/A Software and algorithms FIJI (ImageJ) NIH https://imagej.net/software/fiji/ RRID:SCR_002285 GraphPad Prism 9 GraphPad https://www.graphpad.com/features RRID:SCR_002798 R studio RStudio Team, 2020 https://posit.co/ RRID:SCR_000432 CellPose 2.0 CellPose http://www.cellpose.org/ RRID:SCR_021716 Other 48 EHM multi-well plate TM5 Myriamed GmbH Cat# 50-0105-0010 Method Details Generation of MYOrganoids MYOrganoids from iPSCs were generated adapting the protocol described for engineered heart tissue (Tiburcy et al. 2020). First, iPSC were induced with doxycycline 200 ng/µl for 24 hours to induce the expression of MyoD and Baf60c. The day after, 1.25 x 10 6 iPSC-committed were resuspended in 77 µl of growth media (SKM02, AMSbiokit) supplemented with hES cell Recovery (Stemgent) and molded in hydrogel composed by 40 µl of Bovine Collagen solution 6mg/ml (Sigma-Aldrich), 17.8 µl of Matrigel Growth Factor reduced (GFR, Corning) 10% v/v 3), 40µl 2X DMEM (Gibco) 4) and 5.2 µl of NaOH 0.05 N). For the generation of MYOrganoids including fibroblasts, 1.25 x 10 5 (ratio 1:10) fibroblasts were included in the iPSC-committed mix during hydrogel preparation. 180µl of hydrogel was casted into 48-well plate TM5 MyrPlate (Myriamed), containing in each well a pair of flexible poles (static stretchers) that support the growth of the engineered tissue in a ring shape. After 1 hour of polymerization at 37°C, growth media (SKM02, AMSbiokit) was added for 24 hours. At day 2 of the 3D, growth media (SKM02, AMSbiokit) was replaced by differentiation media (SKM03plus, AMSbiokit) and changed every day until day 14. Muscle force analysis Functional analyses were carried out at day 14 after 3D casting. Contraction experiments were performed using the MyoDynamics Muscle Strip System 840 MD (Danish Myo Technology A/S) and CS4 stimulator (Danish Myo Technology A/S). All functional analysis were performed at 37°C, 5% CO 2 95% O 2 , in Tyrode’s solution supplemented with 25 mM NaHCO 3 . Optimal muscle length was determined by gradually stretching the muscle until 1.0 mN of passive tension registered. Functional tests were performed under isometric and eccentric conditions. MYOrganoids were electrically stimulated with 250 pulses of 30V, 4 ms width at the 125 Hz of frequency for both isometric and eccentric contractions. For eccentric analysis, MYOrganoids were 1 mm stretched at the 6.5 mm/s speed during the muscular contraction. Data collection and analysis was done by PowerLab device and LabChart software (ADInstruments, New Zealand) respectively. Each artificial tissue was subjected to 1 isometric contraction, 10 eccentric contractions and 1 isometric contraction. Fatigue is represented as percentage drop force between the first and the last isometric contraction. Where indicated, force is indirectly normalized for the CSA (Cross Section Area) calculated as muscle force (mN) x Lo (mm) x density (mg/mm3)/weight (mg) and expressed as mN/mm2. Engineered muscle density is experimentally determined as 2.089 mg/mm3. Immunofluorescence MYOrganoids that did not undergo functional analysis, have been analyzed for immunehistofluorescence. Briefly, the artificial tissues are fixed if 4% methanol-free paraformaldehyde (PFA) overnight at day 14. For whole mount staining, fixed MYOrganoids were permeabilized, stained and cleared with the MACS clearing kit (Miltenyi) accordingly to manufacturer’s instructions. Whole mount-stained organoids are then imaged with confocal microscope (LEICA STED SP8) at 10X magnification. For staining on transversal or longitudinal sections, fixed MYOrganoids are dehydrated with a gradient of sucrose (7.5%-30%) over-day and embedded in OCT matrix in plastic mold. After 24 hours, embedded MYOrganoids are processed with the cryostat (LEICA) with 14 µm thick sections. Slices were then dried and fixed again with 4% methanol-free PFA (Invitrogen). Fixed sections are then blocked with serum cocktail (5% Goat serum and 5% Fetal bovine serum), before being stained overnight at +4°C with primary antibody. After that, slices are washed three times in PBS and hybridized with AlexaFluor secondary antibody accordingly to the host species of the first antibody. Stained slides were then covered with Fluoromont + Dapi (SouthernBiotech) and glass slide 1.5H. For imaging, sections are scanned with AxioScan microscope and confocal Leica SP8. Antibodies used are listed in key resources table. For 2D staining, cells are grown on µ-Dish 35 mm (Ibidi) and then fixed in 4% methanol-free PFA for 7 minutes. For membrane and cytoplasmatic stainings, cells are permeabilized with 0.15% Triton X-100 for 10 minutes and then washed with PBS for 5 minutes. For nuclear staining, cells are permeabilized with 0.25% Triton X-100 for 15 minutes. Permeabilized cells are then blocked with serum cocktail (5% Goat serum and 5% Fetal bovine serum), before being stained overnight at +4°C with primary antibody. Cells are then washed three times with PBS for 5 minutes and then hybridized with AlexaFluor secondary antibody according to the host species of the first antibody. After three washing of 5 minutes in PBS, nuclei are stained for 10 minutes with Hoechst 33342 (Invitrogen) at the final dilution of 2 µg/ml. For imaging, sections are scanned confocal Leica SP8. Antibodies used are listed in key resources table. Electron Microscopy studies Electron microscopy analysis was prospectively performed on MYOtissue specimens that were fixed with glutaraldehyde (2.5%, pH 7.4), post fixed with osmium tetroxide (2%), dehydrated in a graded series of ethanol ranging from 30% to absolute solution and embedded in resin (EMBed-812, Electron Microscopy Sciences, USA). 80 nm thick sections from at least four blocks from Ctr iPSC-derived MYOrganoids in presence or absence of Ctr fibroblasts were stained with uranyl acetate and lead citrate. The grids were observed using a “JEOL 1400 Flash” electron microscope (120 kV) and were photo documented using a Xarosa camera (Soft Imaging System, France). Images covering whole longitudinal sections were assessed and representative images were used. Images analysis Myotubes alignment FIJI and CellPose were used for image analysis. Myotube alignment was determined by angle measurement and by myotube circularity from cross-section cuts of MYOrganoids. Angles between two myotubes were measured with the “angle tool” function in FIJI, by drawing two lines perpendicular to two adjacent myotubes membrane. Myotubes circularity was determined by custom FIJI script. Briefly, myotubes cross section area was first segmented by pre-trained Cellpose2 cyto2 model (Stringer et al. 2021) and then converted into Regions of Interest (ROIs) by Labels_To_Rois.py plugin (Waisman et al. 2021) for subsequent quantification on FIJI (Ferret diameters X and Y). After that, ratio between Feret’s diameter on axis X and axis Y was assessed. At least 4 frames for 3 biological replicates have been analyzed. Maturation and fusion index Maturation index was assessed counting nuclei contained in striated myotubes identified by sarcomeric alpha actinin SAA staining in FIJI software and normalized for the total number of nuclei contained in myotubes. Fusion index has been calculated counting myotubes myosin heavy chain (MyHC) positive containing 1, 2, 3 or more than 3 nuclei as a percentage of the total number of nuclei. At least 4 frames for 3 biological replicates have been analyzed. Z-disk length analysis Z-disk length has been assessed by drawing straight lines in FIJI and by measuring the length in at least 10 mature myotubes per condition. Fibronectin and phospo-SMAD3 quantification Fibronectin protein has been quantified as “IntDen” which is the product of Area and Mean Gray Value on FIJI software. Phospho-SMAD3 has been measured as a percentage of positive nuclei. At least 4 frames for 3 biological replicates have been analyzed. Dystrophin, α-dystroglycan and β-dystroglycan quantification MYOtissue cross sections were stained sarcomeric alpha-actinin (SAA) for myotubes cytosol labeling. The Cellpose2 cyto2 model 10 was fine-tuned on manually myofiber-labeled images based on SAA staining (hyperparameters: n_epochs=200, learning_rate=0.05, weight_decay=0.0001). The labeled dataset used in fine-tuning was prepared in such a way that the model can simultaneously segment myotubes and ignore low-quality staining areas. Fine-tuned models were then used to extract myotubes masks. Reconstruction of myotubes masks was done using the cellpose package (Stringer et al. 2021). Reconstructed masks were then converted into Regions of Interest (ROIs) for subsequent quantification (each ROI corresponds to an individual myofiber) using the Labels_To_Rois.py FIJI plugin (Waisman et al. 2021). The generated ROIs were used for subsequent quantification using FIJI macro. Positive ROIs for dystrophin / alpha-dystroglycan / beta-dystroglycan signal were counted and represented as a percentage of the total ROIs of one image. Gene expression analysis For gene expression analysis, RNA was isolated from MYOrganoids by RNeasy micro kit (QIAGEN) accordingly to manufacturer’s instructions, controlled and quantify by Nanodrop. Around 0.5-1µg of RNA was retro-transcribed to cDNA thanks to the RevertAid H Minus First Strand cDNA Synthesis Kit (Invitrogen). Droplet digital PCR was performed to assess the expression of myogenic factors (MyoD, MYH2, MYH7, MCK, PAX7), of fibrotic markers (COL1A, FN1) and µDystrophin, thanks to the QX200™ ddPCR™ EvaGreen Supermix (Biorad). Gene expression results in copy/µl are represented as fold change to the expression of MyoD normalized for GAPDH housekeeping gene. Primers used are listed in table S1. Biomarker analysis from conditioned media Media was collected just before muscle functional analysis after being in culture for 24 hours (media “before”). After the exercise, MYOrganoids were kept in culture with fresh media for other 24 hours (media “after”). The media was then analyzed for assessment of cytokines concentration. Secretome analysis was performed using the MILLIPLEX® Multiplex Assays Using Luminex® Technology (Millipore), according to manufacturer’s instructions. Briefly, media was analyzed for determining the concentration of tumor necrosis factor-alpha (TNFα), interleukin 6 (IL6) and interleukin 8 (IL8). AAV production and MYOrganoids infection Recombinant AAVs were produced as previously described (Bourg et al. 2022) using AAV9 serotype. Purification was performed using affinity chromatography and titration was done by ddPCR using transgene-specific primers. For optimization of infection, an AAV9-CMV-GFP construct was used. The micro-dystrophin transgene used in the study, under the control of spc512 promoter, was an optimized version of the construct used for GENETHON’s preclinical investigation and clinical trial (Le Guiner et al. 2017b), with deletion from spectrin-like repeats 4 to 23 and full C-terminal truncation, here referred as µDys. Infection in MYOrganoids was performed by delivering the AAV9 particles diluted into the differentiation media at day 7, at two different doses: 1^9 vg/MYOtissue (low dose) and 5^10 vg/MYOtissue (high dose). Media was replaced after 24 hours from the infection and changed daily until day 14. Viral copy number analysis Viral DNA was extracted from mature MYOrganoids by NucleoMag Pathogen kit (Macherey Nagel) using Kingfisher instrument (Thermofisher). DNA yield and purity was assessed by Nanodrop; VCN (viral copy number) was identified by droplet digital PCR using supermix for probe (Biorad). Results are shown as copy number variation using P0 as reference DNA. Primers used are listed in Table S1. Capillary western blot analysis MYOrganoids proteins were extracted in RIPA buffer supplemented with Protease Inhibitor Cocktail EDTA-free (Roche) and Benzonase by homogenization. Total proteins were then quantified by BCA method, thanks to the Pierce 660 protein assay kit (Invitrogen) according to manufacturer’s instructions. Protein detection has been performed by capillary western blot, thanks to the JESS protein simple (Bio-techne), according to manufacturer’s directions. Dystrophin detection (both full-length and µDystrophin) has been performed by the antibody DysB (NCL-DYSB, Leica, 1:20) and its expression has been quantified by total protein normalization. RNA sequencing and transcriptomic analysis The RNA quality of samples was verified using the Bioanalyzer 2100 (Agilent) and Qubit fluorometric quantification (ThermoFisher Scientific). The samples that had an RNA integrity number higher than 9 were used for RNA sequencing (Genewiz). The Stranded Total RNA Library Prep Kit (Illumina) was used to create the sequencing libraries, which were sequenced following the Illumina protocol on the NovaSeq instrument (Illumina), resulting in approximately 20 million paired-end reads per library. The paired-end reads were filtered and subjected to quality control using fastp (Chen et al. 2018). They were then mapped to the GRCh38/hg38 genome using HISAT2 (Kim, Langmead, and Salzberg 2015) count tables were generated using htseq-count (Love, Huber, and Anders 2014). Differentially expressed genes (DEGs) were identified using the DESeq2 R package with p value adjusted by Benjamin-Hochberg procedure less than 0.05. Pathway analysis was carried out in R-Studio (version 4.0.3) using either over-representation methods with ReactomePA (Yu and He 2016) or functional class scoring with Gene Set Enrichment Analysis (Subramanian et al. 2005; Mootha et al. 2003). 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Sci Rep 11 Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15 Subramanian A et al (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102:15545–15550 Mootha VK et al (2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34:267–273 Additional Declarations There is NO Competing Interest. Supplementary Files OKSuppmatPalmierietal.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4270736","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":295823893,"identity":"0fb46158-c1fb-42a1-bddc-a4748f04ea13","order_by":0,"name":"Sonia 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Myologie","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"","lastName":"Bigot","suffix":""},{"id":295823902,"identity":"87f4333c-0063-440f-84a5-1bf21a71b31b","order_by":9,"name":"David israeli","email":"","orcid":"https://orcid.org/0000-0003-2762-2195","institution":"Genethon (France)","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"israeli","suffix":""},{"id":295823903,"identity":"42abb043-2b02-4f98-bfcb-bc0f752ee091","order_by":10,"name":"Isabelle Richard","email":"","orcid":"","institution":"Genethon","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Richard","suffix":""}],"badges":[],"createdAt":"2024-04-15 15:40:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4270736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4270736/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74588293,"identity":"27ff7a04-d471-4899-b687-6548bc7f1869","added_by":"auto","created_at":"2025-01-23 17:10:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":907814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGeneration of iPSC-derived MYOrganoids and impact of fibroblast inclusion on muscle organization.\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e(A\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eScheme of the protocol used to generate muscle artificial tissues (MYOrganoids) from iPSC committed towards the myogenic lineage by 24h treatment with doxycycline for inducible expression of Myod and BAF60C transgenes (iPSC\u003c/em\u003e\u003csup\u003e\u003cem\u003eBM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e). The casting procedure included: committed iPSC, fibroblasts when indicated (+/-fibro) and a collagen-based scaffold, within a 48-well plate equipped with silicon pillars. After 2 days in growth medium, the 3D structures were shifted to differentiation medium until day 14 for histological analysis. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Condensation kinetics of MYOrganoids +/-fibro. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Whole-mount staining of MYOrganoids with SARCOMERIC α-ACTININ (SAA) and 3D reconstruction of the ring-shaped constructs using confocal imaging. Scale bar: 1 mm. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRepresentative longitudinal and cross sections of MYOrganoids +/- fibroblasts, immunostained for Myosin Heavy Chain\u003c/em\u003e \u003cem\u003e(MYHC) and VIMENTIN or for wheat germ agglutinin (WGA). Nuclei were visualized with DAPI. Scale bars 200 µm (left panel) and 10 µm (middle and right panel). Arrows indicate fibroblasts recruited adjacently to the muscle fibers; α is the angle formed between myotubes; lines indicate aligned myotubes, crosses represent X/Y myotubes diameters. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eThe alignment was calculated based on the angle (α) formed between myotubes (α close to 0 corresponds to aligned myotubes, while far from 0 corresponds to not aligned myotubes), while circularity from X/Y myotubes diameters ratio (ratio 1 circular, far from 1 not circular). Data were collected from 3 independent experiments with at least 3 replicates. N=35. Unpaired two-tailed t-test was used (***p ≤ 0.001, ****p ≤ 0.0001).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/ac2076543c5d597ac664de73.png"},{"id":74588295,"identity":"1c5fb302-7099-4ec2-810e-b85c0d4681c2","added_by":"auto","created_at":"2025-01-23 17:10:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1214442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eStructural and functional maturation in fibroblast-including MYOrganoids. (A) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRepresentative transversal sections stained for DYSTROPHIN (Dys). Scale bar: 40 µm. Nuclei were visualized with DAPI. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Representative longitudinal sections of MYOrganoids +/- fibroblasts (fibro), immunostained for SARCOMERIC α-ACTININ (SAA). Scale bars: 40 µm, enlargement 10 µm. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Transmission electron microscopy images showing sarcomeric structures. Orange arrows: Z-lines. Scale bar: 500 nm. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) DYSTROPHIN staining quantification represented as mean intensity fluorescence and expressed as fold change to the negative control (sections stained without first antibody). N=2 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Quantification of maturation index was performed by calculating the nuclei inside striated myofibers visualized by SAA staining as a percentage of the total number of myofibers. N=3-5 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Quantification of fusion index calculated as % MYOSIN HEAVY CHAIN positive (MYHC+) myotubes containing different numbers of nuclei (n) as depicted. N=3 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Gene expression analysis of MCK, MYH2 and MYH7, reported as gene expression relative to MYOD expressing population. N=2-5 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Width of Z-line visualized by Electron microscopy, in MYOrganoids with and without inclusion of fibroblasts. N=16 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Contractile muscle force analysis of MYOrganoids using a muscle-strip-based organ bath system. Lo, the optimal length used for normalization of force data (see methods). (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eJ\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Representative tetanic traces of MYOrganoids with and without the inclusion of fibroblasts. N=3 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Normalized tetanic force peak in MYOrganoids +/- fibroblasts. N=8-15). Data are presented as means +/- SEM. Unpaired t-test was applied for statistical analysis (*p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/f57baa4e22b696044af2f8b8.png"},{"id":74588291,"identity":"ab19dd01-7377-4411-a872-76502ff0ae08","added_by":"auto","created_at":"2025-01-23 17:10:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1644257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDMD iPSC-MYOrganoids including DMD fibroblasts exacerbate pathogenic hallmarks. (A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eOverview of MYOrganoids generation from control iPSC (Ctr1 and Ctr2), DMD iPSC (DMDdEx45, DMDdEx8-43) and isogenic iPSC (corrDMDdEx8-9 and DMDdEx8-9) including Ctr or dystrophic fibroblasts respectively for histological characterization and gene expression analysis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. (B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Immunostaining of MYOrganoids cross sections for DYSTROPHIN (Dys) or FIBRONECTIN (FN1)/pSMAD3 and SAA in longitudinal sections. Scale bar: 40 µm. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Quantification of % of pSMAD3 positive nuclei in all Ctr and all DMD MYOrganoids. N=3 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Quantification of FIBRONECTIN fluorescence signal in all Ctr and DMD MYOrganoids. N=3\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (E-F)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRT-ddPCR analysis of fibrotic markers Fibronectin 1 (FN1) (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and Collagen 1 (Col1A1) (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) in all Ctr and all DMD iPSC-derived MYOrganoids with or without fibroblast. Isogenic cell lines are represented in a separate graph. N=2-4. Data are presented as mean ± SEM. Unpaired t-test was performed for statistical purposes (*p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.000), ns= not significant.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/cec7b48d5965506a5e96f290.png"},{"id":74588297,"identity":"4fa1cdeb-1b07-4245-9122-094afe149264","added_by":"auto","created_at":"2025-01-23 17:10:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSignificant muscle force loss and reduced endurance in DMD MYOrganoid including dystrophic fibroblasts\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Force drop over 10 repetitions of eccentric (ECC) contractions in all Ctr and all DMD MYOrganoids without (A) or with g fibroblasts (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) N=4-6. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Fatigue index, calculated as % of drop force between two ISO performed before and after 10x repetitions of \u003c/em\u003eECC\u003cem\u003e in all Ctr and all DMD MYOrganoids +/- respective fibroblasts. N=5-10 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Fatigue index, calculated as % of drop force between two ISO performed before and after 10x repetitions of \u003c/em\u003eECC\u003cem\u003e in corrDMDdEx8-9 and DMDdEx8-9 MYOrganoids +/- three Ctr and DMD fibroblasts obtained from different genetic source. N=3-6 Data are presented as mean ± SEM. Unpaired t-test was performed for statistical purposes (*p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.000), ns= not significant.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/397044c5694655a70e63f1a9.png"},{"id":74588299,"identity":"7d910551-fe44-45fa-bca6-bd0642d892fd","added_by":"auto","created_at":"2025-01-23 17:10:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1282265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePartial restoration of DGC members following AAV-mediated delivery of µDys in profibrotic DMD MYOrganoids\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Scheme of AAV9-µDys infection and doses used in DMD iPSC-derived MYOrganoids. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB-C-D\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Evaluation of gene transfer efficiency by Viral copy number (VCN) analysis (N=3) \u0026nbsp;\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, mRNA expression levels (N=2-4) \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eand protein expression analysis by capillary western blot\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ein the representative isogenic iPSC DMD dEx8-9-derived MYOrganoids infected with low and high dose of µDys versus non-infected (-) corrDMDdEx8-9 (N=2-3) \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Histological evaluation of Dystrophin Glycoprotein Complex (DGC) upon infection, by immunostaining for Dystrophin (Dys), α-Dystroglycan (α-DG) and β-Dystroglycan (β\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eDG\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e of Ctr and DMD cross sections of MYOrganoids. Images are relative to the representative isogenic iPSC. Scale bar: 40 µm. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(F\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) DYSTROPHIN (DYS), α-DYSTROGLYCAN (α-DG) and β- DYSTROGLYCAN (β\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eDG\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e quantification in DMDdEx8-9 iPSC-MYOrganoids cross sections and its isogenic corrected control (corr), N=3-6. Data are presented as means +/- SEM. Statistical analysis was performed with an ordinary one-way ANOVA test (*p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant) with multiple comparisons corrected with Tukey’s test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/f90117ccd13d00289fed8db7.png"},{"id":74588298,"identity":"ca9509c0-bcf7-4f37-a221-627d1c1f02e8","added_by":"auto","created_at":"2025-01-23 17:10:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":189559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMuscle strength evaluation and secretome analysis following microdystrophin gene transfer.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Ctr and DMDdEx45 MYOrganoids subjected to muscle force assessment and secretome analysis following AAV-µDys gene transfer.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (B-C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Drop force over 10 repetitions of eccentric (ECC) contractions in isogenic cell lines (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and Ctr1, Ctr2 and DMDdEx45, DMDdEx8-43 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)-derived MYOrganoids receiving low and high doses of µDys. N=6-7 \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Fatigue index in all Ctr and DMD MYOrganoids treated or not with m-Dystrophin at low and high dose. N=5-6 \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eQuantification of TNFα, IL-6 and IL-8 cytokines released in the media collected 24h after eccentric training. N=4-5. Data are presented as mean ± SEM. Statistical analysis was performed with unpaired t-test (*p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001) ns, not significant.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/4767c55d590f087d64cc0096.png"},{"id":74588837,"identity":"6cdf9b46-7491-496b-abb8-f6107132fc2f","added_by":"auto","created_at":"2025-01-23 17:18:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":365731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTranscriptomic analysis in µDys-treated DMD MYOrganoids. (A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Principal component analysis (PCA) of DMD MYOrganoids transcriptomics treated or not with µDystrophin. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Heat map depicting relative transcript levels of differentially expressed genes in DMD MYOrganoids transcriptomics treated or not with µDystrophin (untreated vs DMD_µDys_high). Lower and higher expressions are depicted in blue and red, respectively. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Volcano plot showing the differential gene expression in DMD MYOrganoids treated with a high dose of µDystrophin vs untreated. The data points above the significance threshold (p_adjusted \u0026lt; 0.05, fold2change \u0026gt; 2) are marked in blue (down-regulated) and red (upregulated), and others are marked in gray (not significant). (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Dot plot of the 15 most significant REACTOME pathways from enrichment analysis of all significantly upregulated genes upon µDys_high treatment compared to untreated DMD. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGSEA of RNA-seq from DMD MYOrganoids untreated and treated with high dose µDys. N=4.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/ebeb0823257fc0756107375a.png"},{"id":74589690,"identity":"2f93560f-f671-4cd8-9bef-9f89b8d3c95d","added_by":"auto","created_at":"2025-01-23 17:26:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6831778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/0c993c12-071c-48ca-a501-100dd3c2bdec.pdf"},{"id":74588292,"identity":"8cd3c3d1-c099-4894-bb5d-1a3e42c78135","added_by":"auto","created_at":"2025-01-23 17:10:34","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":459718,"visible":true,"origin":"","legend":"","description":"","filename":"OKSuppmatPalmierietal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4270736/v1/cc31b60708ee2cf8a2ada758.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Disease exacerbation in MYOrganoids derived from Duchenne Muscular Dystrophy iPSC reveals limitations of microdystrophin therapeutic efficacy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuchenne muscular dystrophy (DMD; ONIM: #310200) is an X-linked disorder that affects one in every 5000 male births\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e with no resolutive cure up to date. It is characterized by progressive muscle wasting affecting skeletal muscles primarily and cardiac and respiratory muscles later, thereby causing premature death \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. DMD is caused by genetic mutations in the \u003cem\u003eDMD\u003c/em\u003e gene, leading to the absence of Dystrophin, an essential protein that provides physical support to myofibers by linking them to the extracellular matrix through the Dystrophin Glycoprotein Complex (DGC) \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The lack of Dystrophin results in a series of muscle membrane breakdowns and repairs, leading to subsequent secondary issues like chronic inflammation and fibrosis \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Fibrosis is an excessive deposition of extracellular matrix components like fibronectin and collagen, triggered by overactivation of Transforming Growth Factor beta (TGF-β) and leading to loss of muscle functionality\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Besides being a critical driver of DMD progression, fibrosis also hampers gene therapy efficacy and is therefore paramount to counteract this process that is well-established in patients.\u003c/p\u003e \u003cp\u003eGene therapy using adeno-associated virus (AAV) is currently the most promising treatment for Duchenne muscular dystrophy. Ongoing clinical trials use AAV to deliver short forms of Dystrophin, known as microdystrophin (\u0026micro;Dys), which encodes a truncated but functional protein \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, while the therapeutic effects were unequivocally achieved in DMD animal models \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, the results from clinical trials revealed only partial therapeutic efficacy in terms of gain of muscle function and rarely addressed whether fibrotic activity and signaling were reduced by gene transfer \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These observations confirm the limited translatability of results obtained in animal models to human patients. It appears therefore crucial to develop time and cost-effective high throughput models, mimicking the severity of human DMD pathology, suitable for research investigation and therapeutic screening.\u003c/p\u003e \u003cp\u003eIn this context, \u003cem\u003ein vitro\u003c/em\u003e modeling based on human cells is a valuable option. In particular, the induced pluripotent stem cells (iPSC) technology offers the opportunity to derive an unlimited number of specialized cells from patients for disease modeling and drug screening \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Among the \u003cem\u003ein vitro\u003c/em\u003e cellular models, organoid-like structures are becoming invaluable for disease modeling as the use of 3D cultures and biomaterials allows the reconstitution of tissue architecture and microenvironment that are instrumental for pathophysiological evaluations \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Tissue engineering applications for AAV gene therapy have been exploited mostly in the context of retinopathies \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e while only limitedly explored for muscular disorders. Hence, having human DMD models is of utmost importance to advance gene therapy and provide a platform for predictive screening. Although several \u003cem\u003ein vitro\u003c/em\u003e 3D systems are accessible for modeling Duchenne muscular dystrophy \u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, their throughput use is limited by the long duration, variability, related to the complexity of cellular composition achieved, and lack of disease-specific readouts for muscle function.\u003c/p\u003e \u003cp\u003eHere, we report on the generation of iPSC-derived muscle organoid structure, named hereafter MYOrganoids. We employ and adapt an engineered muscle platform to generate MYOrganoids using a previously reported method for direct iPSC conversion into 2D skeletal muscle cells \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. As a strategy to increase the structural and functional maturation required for pathophysiological studies, we use fibroblasts as they are a major source of connective tissue which is a key regulator of differentiation and muscle structure. Moreover, fibroblasts act as a source of microenvironment cues exerted by their secretory activity and they are therefore regulators of the muscle niche that undergoes pathological remodeling during disease\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e .Here we show that fibroblast inclusion enhances the structural and functional maturation of the muscle cells. In a DMD context, fibroblasts allow exacerbation of phenotypic traits by direct interaction with muscle cells and reveal key hallmarks of DMD such as fibrosis and muscle weakness over repeated contractions.\u003c/p\u003e \u003cp\u003eOur study also evaluates for the first time the therapeutic efficacy of AAV-mediated \u0026micro;Dys gene transfer, in engineering muscle tissues, as proof of concept of their suitability for studying disease mechanisms and evaluating potential therapeutics. By using different doses of \u0026micro;Dys in DMD MYOrganoids, we observed a dose-dependent response in restoring muscle function while only a partial effect at the level of membrane stability and fibrotic signature in DMD muscles. Our findings indicate that patient-derived MYOrganoids, whose pathogenic traits are exacerbated, are suitable for studying the fibrotic process orchestrated by either muscle or fibroblast population and its interplay with gene transfer approaches. Our system has therefore the potential to identify molecular mechanisms driving the dystrophic process and accelerate the identification of effective therapeutics for DMD.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeneration of structurally organized 3D human MYOrganoids by direct conversion of iPSC and inclusion of fibroblasts\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMYOrganoids were generated from human iPSC committed to differentiating into the myogenic lineage by inducible expression of MyoD and BAF60C \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (referred to as iPSC\u003csup\u003eBM\u003c/sup\u003e.) that can directly generate myotubes. MYOrganoids were prepared starting from iPSC\u003csup\u003eBM\u003c/sup\u003e after one day from the induction of myogenic genes. The casting procedure was performed through adaptation of an engineered muscle system \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e which results in the growth of the tissue in a ring format supported by two flexible silicon stretchers. The 3D cultures were kept for 2 days in growth medium, afterwards medium was replaced for differentiation for another 12 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The differentiation protocol was optimized from the conditions previously reported \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e using myogenic commercial media that ensured the highest expression of myogenic markers and myogenic differentiation in monolayer conditions (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B\u003c/b\u003e). Since cellular heterogenicity, especially of mesenchymal origin, is important for muscle formation \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, we included human fibroblasts during the casting procedure, to assess whether this would affect muscle organization. For this aim, casting was performed using iPSC\u003csup\u003eBM\u003c/sup\u003e cells in the presence or absence of human fibroblasts. Achieving alignment and differentiation simultaneously necessitates a delicate balance between fibroblasts and muscle cells (as noted by N. Rao et al., 2013). To replicate physiological conditions accurately, we incorporated a fibroblast concentration that mirrors the stromal population detectable through single cell and single nuclei-RNA seq analysis of muscles \u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We found that including 10% fibroblasts, accelerated the condensation and growth over time of the muscle rings into a compact structure 0.8 mm long and 1 mm thick at day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e By performing immunofluorescence in whole-mount tissues for sarcomeric α-actinin (SAA), we could detect an enrichment of SAA-positive myotubes throughout the ring-shaped micro-tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We then aimed to assess the impact of fibroblast inclusion on muscle structure. Since the organization of muscle cells within the ECM plays a key role in fusion and maturation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, both alignment of myotube and circularity, a consequence of their parallelism, were evaluated. Staining for myosin heavy chain (MyHC, myotube marker) and vimentin (fibroblasts marker) on longitudinal sections showed fibroblast recruitment near muscle fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Myotubes alignment was determined by measuring the angles in between myotubes \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and showed a significant decrease towards 0 degrees upon fibroblast inclusion, which indicates parallelism while, in the MYOrganoids without fibroblasts, we detected a disordered pattern of muscular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Additionally, the circularity of myofibers was measured from transversal sections stained for the membrane marker wheat germ agglutinin (WGA), using the ratio between X and Y Feret diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). MYOrganoids including fibroblasts had an improved circularity (ratio closer to 1) when compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Improved myotube circularity and alignment as shown, indicate that fibroblast incorporation during the casting procedure guides skeletal cell orientation providing structural support for MYOrganoids, a prerequisite for maturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIncreased structural and functional maturation of fibroblast-including MYOrganoids\u003c/h2\u003e \u003cp\u003eSince muscle maturation is strictly dependent on the internal myofiber organization, we evaluated the differentiation of our 3D MYOrganoids by looking at the sarcomere structure. Transversal and longitudinal sections were used to monitor dystrophin (DYS) expression at the sarcolemma \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and sarcomeric α-actinin (SAA) localization for assessment of the striation pattern typical of mature myotubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Dystrophin was properly localized to the muscle membrane of myotubes from MYOrganoids including fibroblasts and was significantly more expressed than MYOrganoids without fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Remarkably, the maturation index, reported as a percentage of the number of nuclei included in striated myotubes, was significantly superior in MYOrganoids including fibroblasts as compared to MYOrganoids without fibroblasts which appear very disorganized with a rare appearance of striations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Increased maturation of myotubes within MYOrganoids including fibroblast was also supported by analysis of the fusion index, indicating a significantly higher percentage of multinucleated myotubes (\u0026gt;\u0026thinsp;3 nuclei) and a lower percentage of mononucleated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This evidence highlights the positive role of fibroblasts in the maturation process through fusion and multinucleation. The proper sarcomeric organization was also confirmed by electron microscopy where we could detect longer, properly formed Z patterning and the presence of I and A bands along with an overall increase of sarcomeric density and alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consistently, MYOrganoids containing fibroblasts showed wider Z-line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), index of higher maturation of sarcomeres \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. We further performed gene expression analysis for terminal differentiation markers such as muscle creatine kinase (\u003cem\u003eMCK\u003c/em\u003e), myosin heavy chain (\u003cem\u003eMYH\u003c/em\u003e) isoforms, such as \u003cem\u003eMYH2\u003c/em\u003e, representative of fast adult fiber type, and \u003cem\u003eMYH7\u003c/em\u003e, as a slow fibers marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Higher expression of all genes in MYOrganoids containing fibroblasts confirms the acquisition of a more mature state, compared to MYOrganoids without fibroblasts.\u003c/p\u003e \u003cp\u003eWe then assessed whether our MYOrganoids were functional by evaluating their physiological response to contraction stimulations, using a muscle organ bath system based on electrical pacing \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. To evaluate muscle force, MYOrganoids were transferred to the muscle strip chamber and stretched until the optimal length (Lo) for functional analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Isometric force analysis revealed significantly higher tetanic force in MYOrganoids containing fibroblasts compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Values were then normalized for the cross-sectional area (CSA) using the weight and optimal length of contraction established for each MYOrganoid \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and expressed as specific tetanic force (mN/mm\u003csup\u003e2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). In particular, the highest force with fibroblasts had peak values ranging from 0.3 to 0.5 mN versus 0.1 to 0.2 mN in MYOrganoids without fibroblasts after normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). These data demonstrated that MYOrganoids plus fibroblasts have an improved structural organization and functional maturation that enables force contraction studies by electrical pacing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInclusion of fibroblasts in DMD iPSC-derived MYOrganoids leads to increased muscle fatigue and pro-fibrotic signature\u003c/h3\u003e\n\u003cp\u003eThe improved muscle organization and functional maturation shown by MYOrganoids including fibroblasts, prompted us to exploit fibroblast features in disease modeling for DMD, where their role in disease progression is well known \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We incorporated DMD fibroblasts to recapitulate the pathogenic microenvironment arising from their profibrotic activity exerted by tissue remodeling and matrix deposition \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. For that purpose, we used three DMD iPSC with different \u003cem\u003eDMD\u003c/em\u003e mutations, a deletion of exon 45 (DMDdEx45), a deletion of exons 8\u0026ndash;43 (DMDdEx8-43) and a deletion of exons 8\u0026ndash;9 (DMDdEx8-9) with their isogenic control, the DMD dEx6-9 iPSC corrected to restore dystrophin expression \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e (hereafter called IsoCTR). We additionally used two control iPSC lines derived from healthy patients (CTR1, CTR2). Control and DMD MYOrganoids were generated from control and DMD iPSC with healthy or DMD human immortalized fibroblasts for functional and transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The myogenic differentiation ability of both CTR and DMD iPSC lines was first evaluated in 2D cultures. All cell lines showed comparable myogenic potential, as expected using a direct myogenic conversion protocol bypassing any defective developmental steps (\u003cb\u003eFigure S2A\u003c/b\u003e). Histological characterization in MYOrganoids showed the absence of dystrophin protein in DMD MYOrganoids and efficient myogenic differentiation and maturation in CTR and DMD MYOrganoids, as shown by the striated pattern visualized by SAA-stained sections, confirming the positive role of fibroblasts in enhancing maturation also in a DMD context (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo assess whether DMD MYOrganoids display hallmarks of DMD pathophysiology, we evaluated muscle function, which represents one of the most difficult challenges in establishing therapeutic readouts with \u003cem\u003ein vitro\u003c/em\u003e systems. To identify reliable force parameters reflecting the defective DMD muscle performance, MYOrganoids were subjected to isometric contractions to measure tetanic force, and to eccentric contractions, which play a critical role in the disease progression of DMD\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e by triggering the membrane degeneration/regeneration cycles\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e to assess muscle strength and fatiguability. No significant changes were observed in the tetanic isometric force between CTR and DMD MYOrganoids (\u003cb\u003eFigure S2B\u003c/b\u003e), as expected for muscles never been challenged by contractions and with the same myogenic potential, while DMD MYOrganoids showed higher drop force in the eccentric contraction repetitions exclusively when including DMD fibroblasts starting from the 4th eccentric repetition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). On the other side, CTR and DMD MYOrganoids not including fibroblasts, show significant differences in drop force just at the tenth repetition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To accurately quantify muscle fatigue, we calculated the fatigue index as the drop of force between the isometric contractions performed before and after the 10 repetitions of eccentric exercise. The analysis showed a significantly higher fatigue index in DMD MYOrganoids as compared to CTR MYOrganoids and remarkably, this phenomenon was highly accentuated by the presence of fibroblasts. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. To check whether the impact on disease exacerbation and increase of difference in fatiguability between CTR and DMD MYOtissue was affected by the different genetic backgrounds of the fibroblast source, we employed three additional fibroblast cell lines from either healthy and DMD individuals to generate CTR and DMD MYOrganoids. Muscle force analysis confirmed that the fatigue index in DMD MYOrganoids was significantly higher than CTR MYOrganoids, regardless of the genetic source of either control or DMD fibroblasts. This analysis also confirmed the display of higher and significant muscle fatiguability in fibroblasts-including MYOrganoids (\u003cb\u003eFigure S2C\u003c/b\u003e). Collectively, these data indicate that DMD MYOrganoids including DMD fibroblasts, through their pro-fibrotic activity, display exacerbated loss of muscle resistance and increase in fatiguability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. The finding also demonstrates that eccentric-based drop force evaluation is a meaningful and reliable therapeutic readout, as significant differences were detected between CTR and DMD MYOrganoids including fibroblasts.\u003c/p\u003e \u003cp\u003eWe then performed transcriptomic analysis in the isogenic iPSC lines (IsoCTR and DMDdEx8-9) to better characterize the pathogenic hallmarks introduced by fibroblast incorporation within the organoids. Analysis of significant (p.adj\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and relevantly different transcripts (abs log2FoldChange\u0026thinsp;\u0026gt;\u0026thinsp;1) revealed 4610 differentially expressed genes (DEGs) between DMDdEx8-9 and IsoCTR MYOrganoids, of which 2181 upregulated and 2429 downregulated (\u003cb\u003eFigure S3A-B\u003c/b\u003e). Of those, we identified genes involved in decreased muscular contraction (e.g. muscle system process, muscle contraction, sarcoplasmic reticulum calcium ion transport and actin-myosin filament sliding) as well as reduced energetic molecular process (e.g. oxidative phosphorylation, mitochondrial respiratory chain complex assembly, ATP synthesis coupled electron transport), as depicted by gene ontology biological process (GOBP) enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cb\u003etable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Remarkably, between the upregulated pathways in DMD MYOrganoids, we identified higher collagen fibril organization with an enrichment score of +\u0026thinsp;0.6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This GO analysis was also confirmed by Kyoto encyclopedia of genes and genome (KEGG) enrichment analysis, which showed a downregulation of oxidative phosphorylation and an upregulation of ECM receptor interaction, such as Integrin alpha and beta (\u003cb\u003eFigure S3C\u003c/b\u003e). Interestingly, the analysis of genes involved in the ECM remodeling revealed an upregulation of genes coding for collagens, laminins, metalloproteases and TGF-β related genes such as TGF-B receptor (TGFBR1) and the ncRNA inhibitor of TGF-β (TGFB2-AS1) in DMD MYOrganoids compared to isogenic control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, we found increased levels of latent TGF-β binding proteins (LTBP 2 and 4) in dystrophic MYOrganoids, indicating that TGF-β is activated in the DMD context and contributes to ECM remodeling (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe increased profibrotic signature in DMD iPSC-derived MYOrganoids was then confirmed in all CTR and DMD iPSC-derived MYOrganoids, by evaluation of the expression of two key fibrotic markers, Fibronectin-1 (\u003cem\u003eFN1\u003c/em\u003e) and Collagen-1 (\u003cem\u003eCOL1A1\u003c/em\u003e). Importantly, the differences in the expression of fibrotic markers between CTR and DMD MYOrganoids were significant only when including fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). We further examined, at histological level, the presence of activated TGF-β signaling in DMD organoids by looking at phosphorylated SMAD3 (pSMAD3), the transcriptional effector of the canonical TGF-β pathway. Notably, DMDdEx8-9-derived MYOrganoids showed increased pSMAD3 positive nuclei both in myotubes (MyHC positive staining) and fibroblasts (MyHC negative cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). This observation indicates a crosstalk between fibroblasts and myofibers and supports the potential of using fibroblasts in tissue engineering as a source of ECM and pro-fibrotic cues under pathological conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eJuxtracrine role of fibroblasts in the modeling of DMD phenotype\u003c/h3\u003e\n\u003cp\u003eWe then sought to gain insights into the contribution of fibroblasts in the recapitulation of key pathogenic hallmarks of DMD. Given that fibroblasts are known to primarily act through their secreted molecules \u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003ewe analyzed the secretome of both control (CTR) and DMD MYOrganoids (MyoT\u0026thinsp;+\u0026thinsp;Fibro) and compared it to that of single-cell 3D cultures (Fibro-only and MyoT-only) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Analysis of culture media on day 14 showed elevated levels of secreted TGF-β and Collagen 4 (COL4) in DMD MYOrganoids cocultured with fibroblasts compared to control conditions. No significant differences were observed between control and DMD in Fibro-only and MyoT-only 3D cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These data indicate that DMD MYOrganoids in coculture with fibroblast exhibit an increased release of fibrotic factors.\u003c/p\u003e \u003cp\u003eTo determine whether fibroblasts amplify the fibrotic phenotype through their secretory activity, we collected conditioned media (CM) from control (CTR) and DMD Fibro-only 3D cultures on day 7 of \u003cem\u003ein vitro\u003c/em\u003e growth. We then applied this CM to treat CTR and DMD MyoT-only tissues for additional 7 days, assessing the secretion of fibrotic factors, gene expression, and muscle force (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). ELISA assay on secreted TGF-β and fibronectin 1 (FN1) revealed no difference in MyoT-only 3D cultures treated with the CM from either CTR or DMD Fibro-only tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Congruently, gene expression levels of TGF-β and Col1A1 were similar in CTR and DMD MyoT-only cultures, regardless of the treatment with the CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), while those were higher just in DMD cocultures (MyoT\u0026thinsp;+\u0026thinsp;Fibro) compared to control conditions (\u003cb\u003eFigure S4A\u003c/b\u003e). The lack of effect of the CM indicates that the profibrotic increase seen in DMD MYOrganoids in coculture with fibroblasts occurs by a juxtracrine effect, as it comes from contacts between the two cell types.\u003c/p\u003e \u003cp\u003eWe further evaluated the requirement of fibroblasts-myotubes coculture in disease exacerbation by assessing muscle force analysis following CM treatment, which confirmed no difference in fatiguability in myotube-only 3D cultures treated with control or DMD Fibro-derived CM, unlike higher fatigue index in DMD organoids containing both fibroblasts and myotubes in coculture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Interestingly, MYOrganoids including fibroblasts displayed a higher tetanic force compared to myotubes-only cultures treated or not with fibroblasts CM, also supporting their cell contact-dependent role for enhanced functional maturation (\u003cb\u003eFigure S4B\u003c/b\u003e). Collectively, analysis of secreted protein and force under CM experiments showed that the dystrophic fatiguability and fibrosis traits emerged only when fibroblasts and muscle cells were cocultured, indicating that contact-dependent cellular communication between muscle and fibroblasts is required to reveal the DMD traits of profibrotic secretion and fatiguability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAAV-microdystrophin gene transfer rescues muscle resistance while partially restoring Dystrophin Glycoprotein Complex components in DMD MYOrganoids\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs proof of concept that MYOrganoids were suitable as a screening platform for gene therapy, we used AAV-mediated delivery of microdystrophin (\u0026micro;Dys) and assessed its therapeutic efficacy in the DMD context. We used AAV9 capsid for this aim, as this viral serotype was used in recent clinical trials and showed a high transduction rate in patients\u0026rsquo; myofibers \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. We infected the organoids with a codon-optimized \u0026micro;Dys gene (dR4-23, same protein product used in clinical trials \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e) under the control of the muscle-specific spc512 promoter \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e for gene transfer using AAV9 (AAV9-\u0026micro;Dys) in all DMD MYOrganoids iPSC (dEx45, dEx8-43 and dEx8-9 with isogenic control, IsoCTR) and assessed gene transfer efficiency, muscle force analysis and membrane stability assessment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We first optimized the infection conditions using the reporter AAV9-CMV-GFP in CTR MYOrganoids including fibroblasts. Infection was performed on day 7 of the differentiation protocol, diluting AAV particles directly in the medium, and maintained for an additional 7 days (\u003cb\u003eFigures S5A-C\u003c/b\u003e). Optimal low and high AAV9-\u0026micro;Dys doses were established based on previous dosing studies to have intermediate transduction levels at low doses (1E\u0026thinsp;+\u0026thinsp;09 vg/MYOrganoid), and high transduction levels at high doses (5E\u0026thinsp;+\u0026thinsp;10 vg/MYOrganoid) (\u003cb\u003eFigure S5D\u003c/b\u003e). Gene transfer efficiency was evaluated by quantification of viral copy number (VCN) on genomic DNA and by the expression level of the transgene and the encoded protein, showing a clear dose-dependent entry and expression of \u0026micro;Dys in DMD MYOrganoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, S6A-B). As expected by using a muscle-specific promoter, only the muscle cells (MyoT) but not the fibroblasts expressed the transgene after infection of AAV9-uDys in the single populations (\u003cb\u003eFigure S6C\u003c/b\u003e). We then tested whether \u0026micro;Dys affected the contractility and fatigue resistance of the infected organoids. Isometric tetanic force analysis did not reveal any significant changes in DMD MYOrganoids following \u0026micro;Dys delivery compared to not-infected conditions (\u003cb\u003eFigure S6D\u003c/b\u003e), as expected by first-time contracting muscle tissues. We then challenged the muscles with repeated eccentric repetitions, and we observed a dose-dependent attenuation of the drop force in DMD MYOrganoids after \u0026micro;Dys infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Consistently, the fatigue index was greatly reduced in a dose-dependent manner in all DMD MYOrganoids treated with a high dose of \u0026micro;Dys, although reaching significance only for DMDdEx45 and DMDdEx8-9 MYOrganoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). To confirm that the observed effects were specifically due to \u0026micro;Dys expression rather than AAV infection alone, we infected DMD MYOrganoids with a control vector containing a dystrophin fragment lacking a promoter (hereafter called empty vector), which infection did not result in protein expression (\u003cb\u003eFigure S6E-G\u003c/b\u003e). Particularly, the MYOrganoids infected with the empty vector did not show any differences in the fatigue index analysis, at both low and high dose, while we observed a decreased fatigue index in DMD MYOrganoids infected with an AAV containing \u0026micro;Dys (\u003cb\u003eFigure S6H\u003c/b\u003e). Overall, the muscle force data confirmed that ectopic \u0026micro;Dys expression rescues muscle endurance and fatiguability.\u003c/p\u003e \u003cp\u003eBecause dystrophin exerts its biomechanical support by holding the Dystrophin Glycoprotein Complex (DGC) at the membrane, we monitored key components of this complex, such as the transmembrane β-Dystroglycan (β-DG), directly binding Dystrophin, and the extra-cellular α-Dystroglycan (α-DG), whose proper expression and localization are impaired in the absence of dystrophin \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. To this aim, DMD MYOrganoids infected with low and high dose of \u0026micro;Dys, were subjected to histological analysis. Quantification of \u0026micro;Dys showed around 25\u0026ndash;30% of dystrophin-positive myotubes at low dose compared to 85% in the high dose condition in DMD dEx8-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F) and dEx45 and 65% in the DMD dEx8-43 MYOrganoids (\u003cb\u003eFigure S7A-B\u003c/b\u003e). Immunostaining on transversal MYOrganoids showed a dose-dependent yet not complete restoration of β-DG in all DMD iPSC-derived MYOrganoids. Interestingly, even high doses ensuring nearly total Dystrophin transduction in all the DMD MYOrganoids did not significantly restore α-DG (around 10% in DMDdEx8-9 and DMDdEx8-43 and around 30% in DMDdEx45) suggesting a potential therapeutic limitation of \u0026micro;Dys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Importantly, these data demonstrate the limited restoration of Dystrophin-associated components to the sarcolemma after successful \u0026micro;Dys gene transfer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimal microdystrophin gene transfer partially corrects the transcriptomic profile of DMD MYOrganoids and does not rescue the profibrotic signature\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine the effect of microdystrophin (\u0026micro;Dys) on the signaling pathways and molecular processes characteristics of dystrophic pathology, we performed bulk RNA-seq analysis comparing the corrected DMD dEx8-9 (IsoCTR) and DMDdEx8-9 MYOrganoids untreated and treated with a high dose of AAV9-\u0026micro;Dys (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) of the top 10000 genes with the highest variance showed a distinct transcriptomic profile of DMD organoids treated with \u0026micro;Dys (dEx8-9\u0026thinsp;+\u0026thinsp;\u0026micro;Dys high) and untreated (dEx8-9) compared to isogenic controls (IsoCTR) (\u003cb\u003eFigure S8A\u003c/b\u003e). To evaluate the effect of \u0026micro;Dys in restoring key pathways involved in DMD, we firstly compared the transcriptomic profile of \u0026micro;Dys-treated DMD organoids to untreated DMD organoids (dEx8-9 \u0026micro;Dys vs dEx8-9), and then to the isogenic control (dEx8-9 \u0026micro;Dys vs IsoCTR). Particularly, \u0026micro;Dys-treated DMD organoids showed a total of 327 DEGs, of which 154 upregulated and 173 downregulated, compared to untreated organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cb\u003eTable S2\u003c/b\u003e). Of those 327 DEGs, 52% (170/327) are in common with the DEGs that were found dysregulated in DMD organoids when compared to the isogenic control (\u003cb\u003eFigure S8B\u003c/b\u003e). Therefore, we performed the gene ontology enrichment analysis of the common category to see the effect of \u0026micro;Dys on the DMD-dysregulated pathways. This analysis revealed increased expression of genes involved in the microtubule organization (GOBP: Microtubule organizing center organization NES\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.31), cytoskeleton organization (GOBP: Protein localization to cytoskeleton NES\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.99), and a decreased expression of genes involved in the inflammation (GOBP: Regulation of inflammatory response, NES = -1.45) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe then analyzed the DEGs between the treated organoids and the isogenic control to check the transcriptomic signature not restored by \u0026micro;Dys (dEx8-9-\u0026micro;Dys vs IsoCTR). Particularly, \u0026micro;Dys-treated organoids present 7818 DEGs (4004 upregulated and 3814 downregulated) when compared to the corrected isogenic organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Gene ontology analysis of the DEGs between \u0026micro;Dys-treated DMD organoids and IsoCTR organoids revealed the pathways that are not restored by the gene transfer. Particularly, treated organoids showed persistent downregulation of genes involved in calcium transport (GOBP: Sarcoplasmic reticulum calcium ion transport, NES = -2.15), oxidative phosphorylation (GOBP: Oxidative phosphorylation, NES = -2.29) and upregulation of TGFβ signaling (Hallmark TGFβ signaling, NES\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.57), when compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cb\u003eTable S3\u003c/b\u003e). Consistently, a clustered heatmap of all 1716 most significant differentially expressed genes (DEGs) further highlighted the transcriptomic differences between control and DMD organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eAnalysis of Z-score of fibrotic genes among the three conditions (IsoCTR, DMDdEx8-9 and DMDdEx8-9 treated with \u0026micro;Dys) revealed a persistent but variegated profibrotic signature. In particular, we revealed a slight decrease in the expression of ADAMs (i.e. ADAM17, ADAMTS7, ADAMTS16), collagen genes (e.g. COL11A1, COL4A6) and the TGF-β receptor 1 gene (TGFBR1) in \u0026micro;Dys-treated DMD organoids, when compared to the untreated condition, although still significantly different from isogenic control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cb\u003eright panel).\u003c/b\u003e Additionally, other fibrotic genes, like \u003cem\u003eFN1\u003c/em\u003e and \u003cem\u003eCOL1A\u003c/em\u003e1, Integrins or cytokines (e.g.IL1A), showed no expression changes compared to untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cb\u003eright panel\u003c/b\u003e). Further validation of the expression of \u003cem\u003eCOL1A1\u003c/em\u003e, \u003cem\u003eFN1\u003c/em\u003e, not decreased by the \u0026micro;Dys treatment, and of \u003cem\u003eTGF-β\u003c/em\u003e, partially downregulated, confirmed the persistent expression of those genes in DMD-treated MYOrganoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). To understand the mechanisms behind the unresolved fibrotic process, we evaluated the reduction in TGF-β pathway activation within both cell subpopulations in DMD MYOrganoids following \u0026micro;Dys treatment. Histological staining revealed a significant decrease in pSMAD3-positive nuclei within the skeletal muscle subpopulation of \u0026micro;Dys-treated organoids, whereas no difference was observed in fibroblasts after \u0026micro;Dys treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This result suggests a cell-autonomous correction of this signaling within the muscle population receiving \u0026micro;Dys, but no paracrine effect on fibroblast activity. The persistence of activated TGF-β signaling in the fibroblast subpopulation after \u0026micro;Dys gene transfer (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) can therefore explain the uncorrected profibrotic signature and poses the basis for future investigation aimed at improving or driving beneficial cell-cell communication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we present a novel human-relevant model that simulates key traits of advanced stages, enabling the interrogation of gene replacement effect on muscle function and fibrosis, and facilitating the identification of new therapeutic avenues.\u003c/p\u003e \u003cp\u003eWe report the generation of 3D-engineered muscles called MYOrganoids, composed of a homogeneous population of iPSC-derived skeletal muscle cells \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and fibroblasts, major players in muscle tissue organization and microenvironment regulation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We proved that fibroblast inclusion in the MYOrganoids results in improved structural maturation that allows high functional performance under muscle force evaluation, while also eliciting a fibrotic signature in a DMD context.\u003c/p\u003e \u003cp\u003eImportantly, fibroblast incorporation was pivotal in exacerbating DMD phenotype and revealing fatiguability, which is a top hallmark for DMD muscle function, thereby enabling its unprecedented use as therapeutic readouts in vitro. This is important considering that the capacity to evaluate muscle function in a high throughput manner remains limited, further hindering progress in testing or developing effective treatments. As such, while several remarkable studies reported on muscle force in 3D models \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, evaluating muscle function still presents challenges due to the complexity of identifying disease-specific force parameters and the variability of \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e \u003cp\u003eFurthermore, our study sheds new light on the mechanism underlying the contribution of fibroblasts to the modeling of DMD phenotype. We showed that direct interaction of DMD fibroblast with muscle cells is essential to reproducing key pathogenic hallmarks, such as a profibrotic signature and increased muscle fatigue in DMD MYOrganoids. Interestingly, fibroblast role in the recapitulation of DMD hallmarks was not exerted by their profibrotic secretory activity per se but required direct interaction with iPSC-derived muscle cells. As such, fibroblasts in a DMD context showed higher secretion of fibrotic factors like TGF-β, fibronectin, and collagen through cell-contact signaling, suggesting that cell communications between the two cell populations, fibroblasts and muscle fibers, is crucial to reveal pathogenic traits. One possible hypothesis is that fibroblasts secretory activity is enhanced by alterations in cell communications with dystrophin-deficient muscle cells whose membrane stability is impaired, thereby causing increased matrix deposition and susceptibility to contraction-induced damage and fatigue.\u003c/p\u003e \u003cp\u003eOverall, the exacerbated severity of DMD MYOrganoids enabled the evaluation of the therapeutic potential of \u0026micro;Dys, currently employed in clinical trials. Consistently with the partial efficacy in the clinics, \u0026micro;Dys \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e delivery in DMD MYOrganoids did not fully rescue DMD phenotype as it led to incomplete restoration of the components of the dystroglycan complex and no reduction in fibrotic genes transcriptome. Notably, we observed a reduction in the TGF-β pathway activation exclusively on muscle cells, while no effect was observed in fibroblasts. This can explain the persistence of ECM protein production and secretion in \u0026micro;Dys treated DMD MYOrganoids, while we observed an effect in the restoration of muscle strength.\u003c/p\u003e \u003cp\u003eOur findings align with previous results showing that \u0026micro;Dys exhibits limited effectiveness in reducing fibrosis in advanced disease stages \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and in more severe models and muscles affected by the disease, such as the diaphragm in the Dba2 \u003cem\u003emdx\u003c/em\u003e model \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. We can speculate that the partial restoration of DAG complex after gene transfer causes mechanical alterations of the membrane that are translated as profibrotic signals. In advanced disease stages, the stiffened ECM perpetuates fibrosis through mechanotransduction, activating fibroblasts to deposit ECM proteins and increasing rigidity. This feedback loop hinders microdystrophin's ability to reverse fibrosis.\u003c/p\u003e \u003cp\u003eUp to now, no other work revealed the impact of \u0026micro;Dys on the fibroblast population of the muscle, revealing the potential of our system in highlighting specific cell-cell communications in response to treatment. Further work is needed to fully understand the role of fibroblasts in the dystrophic process. For example, how the absence of dystrophin in the myotubes is influencing fibroblast activity, and how the reintroduction of \u0026micro;Dys is affecting the composition of the ECM, still needs to be elucidated.\u003c/p\u003e \u003cp\u003eOverall, pro-fibrotic DMD MYOrganoids provide a valuable tool either for the investigation of mechanisms driving dystrophic process and as a human \u003cem\u003ein vitro\u003c/em\u003e counterpart to animal \u003cem\u003ein vivo\u003c/em\u003e preclinical studies, with the potential to unravel therapeutic limitations of current DMD treatments and accelerate the identifications of new treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to Rene Hummel (DMT), Guillaume Tanniou, and Nicolas Guerchet for their invaluable technical support in muscle force evaluation. We also extend our appreciation to J\u0026eacute;r\u0026eacute;mie Cosette for the assistance in imaging and to the histology team for their technical expertise. Finally, we would like to thank Frederique Magdinier for generously providing the DMD iPSC dEx45 and Ctr1 iPSC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Institut National de la Sante et de la Recherche Medicale (INSERM) and by the \u0026ldquo;Association Fran\u0026ccedil;aise contre les Myopathies\u0026rdquo; (AFM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;SA and LPa designed the experiments. LPa performed most of the experiments, including iPSC culture, organoid generation, imaging, and muscle force analysis. LPi performed force analysis, gene expression analysis and western blot experiments. AJ analyzed conditioned media biomarkers and performed secretome analysis. LPa, MM contributed to muscle force analysis. AVH analyzed RNA-seq data. RE and GB performed electron microscopy analysis. AB generated and provided the immortalized human fibroblasts. SA and LPa performed data analysis and figure preparation. SA conceived, supervised the project, and wrote the manuscript. All authors discussed the results.\u0026nbsp;SA, LPa, DI and IR reviewed and edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e All other authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESOURCE AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sonia Albini ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCtr2, corrDMDdEx8-9, DMDdEx8-9 and DMDdEx8-43 hIPS cell lines used in this study are restricted due to MTAs (Ref: MTA 2022 GENETHON - AIM _DMD, MMTA202109-0107, 2021-0296_MTA_CECS).\u003c/p\u003e\n\u003cp\u003eThis study did not generate new unique reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the article and its supplemental information. The RNA sequencing datasets generated in this study can be found in the NCBI Bioproject database (https://www.ncbi.nlm.nih.gov/bioproject) using the access number PRJNA1044721.\u003c/p\u003e\n\u003cp\u003eMicroscopy and functional data reported in this paper will be shared by the lead contact upon request.\u003c/p\u003e\n\u003cp\u003eAny additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e"},{"header":"Experimental Models","content":"\u003cp\u003e\u003cstrong\u003eInduced pluripotent stem cells (iPSCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman iPSCs used in the study were as follows: Ctr1 (AG08C5) derived from healthy fibroblasts (AG08498, Coriell Institute), Ctr2 (M180, CECS), IPSC DMDdEx45 (Coriell, GM25313); DMDdEx8-43 (M202, CECS) DMDdEx8-9 and corrDMDdEx8-9 (CRISPR-mediated skipping of mutated exons dEx6-9, isogenic control) (provided by Doctor E. Olson). iPSCs were maintained using mTeSRplus medium (Stem Cell Technologies) and passaged using ReleSR (Stem Cell Technologies) on Matrigel coated wells (Corning). iPSC engineering and muscle differentiation were performed adapting a transgene-based method previously described (Caputo et al. 2020; Albini et al. 2013). Quality control was performed regularly with Oct-4 and Nanog expression profile and mycoplasma testing and cells were used within 30 passages. Additionally, antibiotic and antimycotic solution (Merk-Millipore) was regularly added to each cell culture media.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll iPSC lines are engineered for transgene-based myotubes differentiation. Briefly, cells were nucleofected (4D Nucleofector, Lonza) with two enhanced versions of piggyBAC (ePB) containing respectively MyoD and BAF60c genes under tetracycline responsive promoter (Caputo L et al 2020). Additionally, iPSCs were regularly selected for the presence of the two ePBs with Puromycin 10 µg/ml and Blasticidin 20 µg/ml (Sigma-Aldrich).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman immortalized fibroblasts\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman immortalized fibroblasts from control (AB1191, AIM) and DMD patient (AB1024, AIM) were generated and obtained from Myobank-AFM of Myology Institute from skin biopsies. Control and DMD human immortalized fibroblasts, used in co-culture with hiPSC in 3D MYOrganoids, were maintained in culture in DMEM High Glucose Lglut supplemented with 20% fetal bovine serum (FBS) and passaged using TrypleE (Gibco).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman primary fibroblasts\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman primary fibroblasts from control (3 lines: 5-8898, 11-1610, 11-1973) and from DMD patients (3 lines, 5-11618, 5-12608, 5-11613) were obtained from DNA bank of Genethon. Fibroblasts were maintained in DMEM high glucose (Gibco) supplemented with 20 ng/ml recombinant human fibroblast growth factor (h-FGF) (Peprotech) and 20% fetal bovine serum (Gibco). Primary fibroblasts were passaged using TrypleE (Gibco)\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd\u003e\n \u003cp\u003eREAGENT / RESOURCE\u003c/p\u003e\n \u003c/td\u003e\u003ctd\u003e\n \u003cp\u003eSOURCE\u003c/p\u003e\n \u003c/td\u003e\u003ctd\u003e\n \u003cp\u003eIDENTIFIER\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal Myosin Heavy Chain (used at 1:10)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDSHB\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# MF20, RRID:AB_2147781\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit polyclonal Vimentin (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eProteintech\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#10366-1-AP, RRID:AB_2273020\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal Dystrophin (used at 1:20)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eLeica Biosystem\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# NCL-DYSB, RRID:AB_563691\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal Fibronectin (used at 1:200)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# F7387, RRID:AB_476988\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit monoclonal pSMAD-3 (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# ab52903, RRID:AB_882596\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal α-Dystroglycan (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMillipore\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 05-298, RRID:AB_309674\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal β-Dystroglycan (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eLeica Biosystems\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# NCL-b-DG, RRID:AB_442043\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit monoclonal alpha-actinin 2 (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 701914, RRID:AB_2688290\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal Oct3/4 (used at 1:200)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# sc-5279, RRID: AB_628051\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit monoclonal to cleaved Caspase-3 (used 1:500)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling technology\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 9664, RRID: AB_ 2070042\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse monoclonal Pax7 (used at 1:100)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDSHB\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# PAX7, RRID:AB_2299243\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGoat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 594\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# A-21125, RRID:AB_2535767\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGoat anti-Rabbit IgG (Heavy chain), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# A27034, RRID:AB_2536097\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGoat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 594\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# A-21135, RRID:AB_2535774\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGoat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 647\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# A-21242, RRID:AB_2535811\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eChemicals, peptides, and recombinant proteins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDoxycycline\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# D9891-5G\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMatrigel (Growth Factor Reduced)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCorning\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 356231\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCollagen 6 mg/ml\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 804622-20ML\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eStemgent hES Cell Cloning \u0026amp; Recovery Supplement\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eOzyme\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# STE01-0014-500\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePuromycin dihydrochloride\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# P8833-10MG\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eBlasticidin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# SBR00022-1ML\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eNaOH\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 221465\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eWheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 488\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# W11261\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eHoechst 33342\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eInvitrogen\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# H3570\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrypLE Express Enzyme\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 12605010\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eReleSR\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eStem Cell Tech\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 100-0484\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eFetal bovine serum\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 16000044\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGoat Serum\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDako\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# X0907\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCritical commercial assays\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A - Immunology Multiplex Assay\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMerck-Millipore\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# HCYTA-60K\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAnti-Mouse Detection Module for Jess, Wes, Peggy Sue or Sally Sue\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eBio-techne\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# DM-002\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRNeasy Micro kit\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eQiagen\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 74404\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eQX200™ ddPCR™ EvaGreen Supermix\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiorad\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 1864034\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eQX200™ ddPCR™ Supermix for Probes\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiorad\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 1863026\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDeposited data\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRNA sequencing data\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eNCBI BioProject database\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePRJNA1044721\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental models: Cell lines\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCtr1 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCoriell\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAG08C5/ AG08498\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCtr2 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCECS\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eM180\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ecorrDMDdEx8-9 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eKyrychenko et al\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMDdEx45 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCoriell\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGM25313\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMDdEx8-9 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eKyrychenko et al\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMDdEx8-43 hIPSC\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCECS\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eM202\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCtr skin fibroblasts immortalized\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAIM\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAB1191\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMD skin fibroblasts immortalized\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAIM\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eAB1024\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts control 1\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e5-8898\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts control 2\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e11-1610\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts control 3\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e11-1973\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts DMD 1\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e5-11618\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts DMD 2\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e5-12608\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary skin fibroblasts DMD 3\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA Bank Genethon\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e5-11613\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOligonucleotides\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ePrimers for ddPCR (see table S5)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eThis paper\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSoftware and algorithms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eFIJI (ImageJ)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eNIH\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttps://imagej.net/software/fiji/\u003c/p\u003e\n \u003cp\u003eRRID:SCR_002285\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad Prism 9\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttps://www.graphpad.com/features\u003c/p\u003e\n \u003cp\u003eRRID:SCR_002798\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eR studio\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eRStudio Team, 2020\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttps://posit.co/\u003c/p\u003e\n \u003cp\u003eRRID:SCR_000432\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellPose 2.0\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellPose\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttp://www.cellpose.org/\u003c/p\u003e\n \u003cp\u003eRRID:SCR_021716\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOther\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003e48 EHM multi-well plate TM5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eMyriamed GmbH\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat# 50-0105-0010\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n"},{"header":"Method Details","content":"\u003cp\u003e\u003cstrong\u003eGeneration of MYOrganoids\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMYOrganoids from iPSCs were generated adapting the protocol described for engineered heart tissue (Tiburcy et al. 2020). First, iPSC were induced with doxycycline 200 ng/µl for 24 hours to induce the expression of MyoD and Baf60c. The day after, 1.25 x 10\u003csup\u003e6\u003c/sup\u003e iPSC-committed were resuspended in 77 µl of growth media (SKM02, AMSbiokit) supplemented with hES cell Recovery (Stemgent) and molded in hydrogel composed by 40 µl of Bovine Collagen solution 6mg/ml (Sigma-Aldrich), 17.8 µl of Matrigel Growth Factor reduced (GFR, Corning) 10% v/v 3), 40µl 2X DMEM (Gibco) 4) and 5.2 µl of NaOH 0.05 N). For the generation of MYOrganoids including fibroblasts, 1.25 x 10\u003csup\u003e5\u003c/sup\u003e (ratio 1:10) fibroblasts were included in the iPSC-committed mix during hydrogel preparation. 180µl of hydrogel was casted into 48-well plate TM5 MyrPlate (Myriamed), containing in each well a pair of flexible poles (static stretchers) that support the growth of the engineered tissue in a ring shape. After 1 hour of polymerization at 37°C, growth media (SKM02, AMSbiokit) was added for 24 hours. At day 2 of the 3D, growth media (SKM02, AMSbiokit) was replaced by differentiation media (SKM03plus, AMSbiokit) and changed every day until day 14.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMuscle force analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFunctional analyses were carried out at day 14 after 3D casting. Contraction experiments were performed using the MyoDynamics Muscle Strip System 840 MD (Danish Myo Technology A/S) and CS4 stimulator (Danish Myo Technology A/S). All functional analysis were performed at 37°C, 5% CO\u003csub\u003e2\u003c/sub\u003e 95% O\u003csub\u003e2\u003c/sub\u003e, in Tyrode’s solution supplemented with 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e. Optimal muscle length was determined by gradually stretching the muscle until 1.0 mN of passive tension registered. Functional tests were performed under isometric and eccentric conditions. MYOrganoids were electrically stimulated with 250 pulses of 30V, 4 ms width at the 125 Hz of frequency for both isometric and eccentric contractions. For eccentric analysis, MYOrganoids were 1 mm stretched at the 6.5 mm/s speed during the muscular contraction. Data collection and analysis was done by PowerLab device and LabChart software (ADInstruments, New Zealand) respectively. Each artificial tissue was subjected to 1 isometric contraction, 10 eccentric contractions and 1 isometric contraction. Fatigue is represented as percentage drop force between the first and the last isometric contraction. Where indicated, force is indirectly normalized for the CSA (Cross Section Area) calculated as muscle force (mN) x Lo (mm) x density (mg/mm3)/weight (mg) and expressed as mN/mm2. Engineered muscle density is experimentally determined as 2.089 mg/mm3.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMYOrganoids that did not undergo functional analysis, have been analyzed for immunehistofluorescence. Briefly, the artificial tissues are fixed if 4% methanol-free paraformaldehyde (PFA) overnight at day 14. For whole mount staining, fixed MYOrganoids were permeabilized, stained and cleared with the MACS clearing kit (Miltenyi) accordingly to manufacturer’s instructions. Whole mount-stained organoids are then imaged with confocal microscope (LEICA STED SP8) at 10X magnification. \u0026nbsp;\u003c/p\u003e\u003cp\u003eFor staining on transversal or longitudinal sections, fixed MYOrganoids are dehydrated with a gradient of sucrose (7.5%-30%) over-day and embedded in OCT matrix in plastic mold. After 24 hours, embedded MYOrganoids are processed with the cryostat (LEICA) with 14 µm thick sections. Slices were then dried and fixed again with 4% methanol-free PFA (Invitrogen). Fixed sections are then blocked with serum cocktail (5% Goat serum and 5% Fetal bovine serum), before being stained overnight at +4°C with primary antibody. After that, slices are washed three times in PBS and hybridized with AlexaFluor secondary antibody accordingly to the host species of the first antibody. Stained slides were then covered with Fluoromont + Dapi (SouthernBiotech) and glass slide 1.5H. For imaging, sections are scanned with AxioScan microscope and confocal Leica SP8. Antibodies used are listed in key resources table.\u0026nbsp;\u003c/p\u003e\u003cp\u003eFor 2D staining, cells are grown on µ-Dish 35 mm (Ibidi) and then fixed in 4% methanol-free PFA for 7 minutes. For membrane and cytoplasmatic stainings, cells are permeabilized with 0.15% Triton X-100 for 10 minutes and then washed with PBS for 5 minutes. For nuclear staining, cells are permeabilized with 0.25% Triton X-100 for 15 minutes. Permeabilized cells are then blocked with serum cocktail (5% Goat serum and 5% Fetal bovine serum), before being stained overnight at +4°C with primary antibody. Cells are then washed three times with PBS for 5 minutes and then hybridized with AlexaFluor secondary antibody according to the host species of the first antibody. After three washing of 5 minutes in PBS, nuclei are stained for 10 minutes with Hoechst 33342 (Invitrogen) at the final dilution of 2 µg/ml. For imaging, sections are scanned confocal Leica SP8. Antibodies used are listed in key resources table.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eElectron Microscopy studies\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eElectron microscopy analysis was prospectively performed on MYOtissue specimens that were fixed with glutaraldehyde (2.5%, pH 7.4), post fixed with osmium tetroxide (2%), dehydrated in a graded series of ethanol ranging from 30% to absolute solution and embedded in resin (EMBed-812, Electron Microscopy Sciences, USA). 80 nm thick sections from at least four blocks from Ctr iPSC-derived MYOrganoids in presence or absence of Ctr fibroblasts were stained with uranyl acetate and lead citrate. The grids were observed using a “JEOL 1400 Flash” electron microscope (120 kV) and were photo documented using a Xarosa camera (Soft Imaging System, France). Images covering whole longitudinal sections were assessed and representative images were used.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eImages analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMyotubes alignment\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFIJI and CellPose were used for image analysis. Myotube alignment was determined by angle measurement and by myotube circularity from cross-section cuts of MYOrganoids. Angles between two myotubes were measured with the “angle tool” function in FIJI, by drawing two lines perpendicular to two adjacent myotubes membrane. Myotubes circularity was determined by custom FIJI script. Briefly, myotubes cross section area was first segmented by pre-trained Cellpose2 cyto2 model (Stringer et al. 2021) and then converted into Regions of Interest (ROIs) by Labels_To_Rois.py plugin (Waisman et al. 2021) for subsequent quantification on FIJI (Ferret diameters X and Y). After that, ratio between Feret’s diameter on axis X and axis Y was assessed. At least 4 frames for 3 biological replicates have been analyzed.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMaturation and fusion index\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMaturation index was assessed counting nuclei contained in striated myotubes identified by sarcomeric alpha actinin SAA staining in FIJI software and normalized for the total number of nuclei contained in myotubes. Fusion index has been calculated counting myotubes myosin heavy chain (MyHC) positive containing 1, 2, 3 or more than 3 nuclei as a percentage of the total number of nuclei. At least 4 frames for 3 biological replicates have been analyzed.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eZ-disk length analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eZ-disk length has been assessed by drawing straight lines in FIJI and by measuring the length in at least 10 mature myotubes per condition.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFibronectin and phospo-SMAD3 quantification\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFibronectin protein has been quantified as “IntDen” which is the product of Area and Mean Gray Value on FIJI software. Phospho-SMAD3 has been measured as a percentage of positive nuclei. At least 4 frames for 3 biological replicates have been analyzed.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDystrophin, α-dystroglycan and β-dystroglycan quantification\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMYOtissue cross sections were stained sarcomeric alpha-actinin (SAA) for myotubes cytosol labeling. The Cellpose2 cyto2 model 10 was fine-tuned on manually myofiber-labeled images based on SAA staining (hyperparameters: n_epochs=200, learning_rate=0.05, weight_decay=0.0001). The labeled dataset used in fine-tuning was prepared in such a way that the model can simultaneously segment myotubes and ignore low-quality staining areas. Fine-tuned models were then used to extract myotubes masks. Reconstruction of myotubes masks was done using the cellpose package (Stringer et al. 2021). Reconstructed masks were then converted into Regions of Interest (ROIs) for subsequent quantification (each ROI corresponds to an individual myofiber) using the Labels_To_Rois.py FIJI plugin (Waisman et al. 2021). The generated ROIs were used for subsequent quantification using FIJI macro. Positive ROIs for dystrophin / alpha-dystroglycan / beta-dystroglycan signal were counted and represented as a percentage of the total ROIs of one image.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGene expression analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFor gene expression analysis, RNA was isolated from MYOrganoids by RNeasy micro kit (QIAGEN) accordingly to manufacturer’s instructions, controlled and quantify by Nanodrop. Around 0.5-1µg of RNA was retro-transcribed to cDNA thanks to the RevertAid H Minus First Strand cDNA Synthesis Kit (Invitrogen). Droplet digital PCR was performed to assess the expression of myogenic factors (MyoD, MYH2, MYH7, MCK, PAX7), of fibrotic markers (COL1A, FN1) and µDystrophin, thanks to the QX200™ ddPCR™ EvaGreen Supermix (Biorad). Gene expression results in copy/µl are represented as fold change to the expression of MyoD normalized for GAPDH housekeeping gene. Primers used are listed in table S1.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBiomarker analysis from conditioned media\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMedia was collected just before muscle functional analysis after being in culture for 24 hours (media “before”). After the exercise, MYOrganoids were kept in culture with fresh media for other 24 hours (media “after”). The media was then analyzed for assessment of cytokines concentration. \u0026nbsp;Secretome analysis was performed using the MILLIPLEX® Multiplex Assays Using Luminex® Technology (Millipore), according to manufacturer’s instructions. Briefly, media was analyzed for determining the concentration of tumor necrosis factor-alpha (TNFα), interleukin 6 (IL6) and interleukin 8 (IL8).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAAV production and MYOrganoids infection\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eRecombinant AAVs were produced as previously described (Bourg et al. 2022) using AAV9 serotype. Purification was performed using affinity chromatography and titration was done by ddPCR using transgene-specific primers. For optimization of infection, an AAV9-CMV-GFP construct was used. The micro-dystrophin transgene used in the study, under the control of spc512 promoter, was an optimized version of the construct used for GENETHON’s preclinical investigation and clinical trial (Le Guiner et al. 2017b), with deletion from spectrin-like repeats 4 to 23 and full C-terminal truncation, here referred as µDys. Infection in MYOrganoids was performed by delivering the AAV9 particles diluted into the differentiation media at day 7, at two different doses: 1^9 vg/MYOtissue (low dose) and 5^10 vg/MYOtissue (high dose). Media was replaced after 24 hours from the infection and changed daily until day 14.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eViral copy number analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eViral DNA was extracted from mature MYOrganoids by NucleoMag Pathogen kit (Macherey Nagel) using Kingfisher instrument (Thermofisher). DNA yield and purity was assessed by Nanodrop; VCN (viral copy number) was identified by droplet digital PCR using supermix for probe (Biorad). Results are shown as copy number variation using P0 as reference DNA. Primers used are listed in Table S1.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCapillary western blot analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMYOrganoids proteins were extracted in RIPA buffer supplemented with Protease Inhibitor Cocktail EDTA-free (Roche) and Benzonase by homogenization. Total proteins were then quantified by BCA method, thanks to the Pierce 660 protein assay kit (Invitrogen) according to manufacturer’s instructions. Protein detection has been performed by capillary western blot, thanks to the JESS protein simple (Bio-techne), according to manufacturer’s directions. Dystrophin detection (both full-length and µDystrophin) has been performed by the antibody DysB (NCL-DYSB, Leica, 1:20) and its expression has been quantified by total protein normalization.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA sequencing and transcriptomic analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe RNA quality of samples was verified using the Bioanalyzer 2100 (Agilent) and Qubit fluorometric quantification (ThermoFisher Scientific). The samples that had an RNA integrity number higher than 9 were used for RNA sequencing (Genewiz). The Stranded Total RNA Library Prep Kit (Illumina) was used to create the sequencing libraries, which were sequenced following the Illumina protocol on the NovaSeq instrument (Illumina), resulting in approximately 20 million paired-end reads per library. The paired-end reads were filtered and subjected to quality control using fastp (Chen et al. 2018). They were then mapped to the GRCh38/hg38 genome using HISAT2 (Kim, Langmead, and Salzberg 2015) count tables were generated using htseq-count (Love, Huber, and Anders 2014). Differentially expressed genes (DEGs) were identified using the DESeq2 R package with p value adjusted by Benjamin-Hochberg procedure less than 0.05. Pathway analysis was carried out in R-Studio (version 4.0.3) using either over-representation methods with ReactomePA (Yu and He 2016) or functional class scoring with Gene Set Enrichment Analysis (Subramanian et al. 2005; Mootha et al. 2003).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAll data were analyzed by GraphPad Prism 9.5.1 software.\u0026nbsp;Parametric tests such as t-tests and ANOVA were used for statistical comparison. To compare two groups, initially F-test was used to compare variances. If there was no difference in variances, statistical comparison was performed using unpaired t-test. To compare multiple groups, we used one-way ANOVA\u0026nbsp;with Tukey’s correction for multiple comparison tests\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eResults were considered significantly different at p \u0026lt; 0.05. \u0026nbsp;Graphs were generated using Graphpad Prism v9 or R version 3.6.2, The figures display the mean ± standard error of the mean.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMendell JR et al (2012) Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol 71:304\u0026ndash;313\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffman EP, Brown RH, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919\u0026ndash;928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErvasti JM, Campbell K (1993) P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 122:809\u0026ndash;823\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney H (1993) L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A 90:3710\u0026ndash;3714\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffman EP, Brown RH, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919\u0026ndash;928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffman EP (2020) The discovery of dystrophin, the protein product of the Duchenne muscular dystrophy gene. FEBS J 287:3879\u0026ndash;3887\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavies KE, Nowak KJ (2006) Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol 7:762\u0026ndash;773\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnnen JP, Verma M, Asakura A (2013) Vascular-targeted therapies for Duchenne muscular dystrophy. Skelet Muscle 3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorter JD et al (2002) A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice. Hum Mol Genet 11:263\u0026ndash;272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmor F et al (2021) Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy. J Cachexia Sarcopenia Muscle 12:677\u0026ndash;693\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214:199\u0026ndash;210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKharraz Y, Guerra J, Pessina P, Serrano AL, Mu\u0026ntilde;oz-C\u0026aacute;noves P (2014) Understanding the process of fibrosis in Duchenne muscular dystrophy. \u003cem\u003eBiomed Res Int\u003c/em\u003e 965631 (2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendell JR et al (2021) duan. \u003cem\u003eMol Ther\u003c/em\u003e 29, 464\u0026ndash;488\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAguti S, Malerba A, Zhou H (2018) The progress of AAV-mediated gene therapy in neuromuscular disorders. Expert Opin Biol Ther 18:681\u0026ndash;693\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan D, Systemic AAV (2018) Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy. 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Nat Genet 34:267\u0026ndash;273\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"iPSC, DMD, fibroblasts, fibrotic, MYOrganoids, AAV, microdystrophin","lastPublishedDoi":"10.21203/rs.3.rs-4270736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4270736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin (\u0026micro;Dys) have shown limited efficacy in patients, contrasting with the favorable outcomes observed in animal models. This discrepancy is partly due to the lack of models that replicate key pathogenic features associated with the severity of the human disease, such as fibrosis and muscle dysfunction. To tackle the translational gap, we develop a human disease model that recapitulates these critical hallmarks of DMD for a more predictive therapeutic investigation. Using a muscle engineering approach, we generate MYOrganoids from iPSC-derived muscle cells co-cultured with fibroblasts that enable functional maturation for muscle force analysis upon contractions. Incorporation of DMD fibroblasts within DMD iPSC-derived muscle cells allows phenotypic exacerbation by unraveling of fibrotic signature and fatiguability through cell-contact-dependent communication. Although \u0026micro;Dys gene transfer partially restores muscle resistance, it fails to fully restore membrane stability and reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our human DMD system, an unparalleled aspect in existing DMD models, and provide the opportunity to explore the underlying mechanisms of dysregulated cellular communication to identify anti-fibrotic strategies empowering gene therapy efficacy.\u003c/p\u003e","manuscriptTitle":"Disease exacerbation in MYOrganoids derived from Duchenne Muscular Dystrophy iPSC reveals limitations of microdystrophin therapeutic efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-23 17:10:29","doi":"10.21203/rs.3.rs-4270736/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da8a91cb-5c43-4b08-87da-d72211dbbc80","owner":[],"postedDate":"January 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31198482,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":31198483,"name":"Biological sciences/Biotechnology/Gene therapy/Targeted gene repair"}],"tags":[],"updatedAt":"2025-03-11T17:40:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-23 17:10:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4270736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4270736","identity":"rs-4270736","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}