{"paper_id":"31435e44-2cf5-4a8d-a2dc-1706a9e842ca","body_text":"Beyond Myoblasts: DUX4 Drives Fibrosis and Myogenic Reprogramming in Mesenchymal Stem Cells | 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 Short Report Beyond Myoblasts: DUX4 Drives Fibrosis and Myogenic Reprogramming in Mesenchymal Stem Cells Olesya SERBINA, Anna SCHWAGER, Ekaterina KISELEVA, Erdem DASHINIMAEV, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9094205/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 We developed a cellular model of mesenchymal stem cells (MSCs) with inducible DUX4 expression (MSC-DUX4) to investigate the potential role of MSCs in facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression was successfully induced, with MSC-DUX4 maintaining the characteristic surface marker profile of MSCs. Unlike myoblasts, which rapidly undergo apoptosis upon DUX4 induction, MSC-DUX4 remained viable although they exhibited increased reactive oxygen species (ROS) accumulation. Transcriptomic analysis revealed broad changes, including strong upregulation of several myogenic genes, suggesting that DUX4 confers a partial myogenic program to MSCs. Indeed, Dox-induced MSC-DUX4 formed myotube-like structures expressing myogenic markers (myogenin, Troponin T, MF20), though fusion efficiency was markedly reduced compared to myoblasts, indicating limited and likely defective myogenic differentiation capacity. In parallel, adipogenic and osteogenic potentials were strongly impaired, as demonstrated by reduced lipid and calcium deposition, altered expression of FABP4 and leptin. Moreover, DUX4-expressing MSCs displayed pro-fibrotic features, including enhanced collagen III/IV and fibronectin, suggesting impaired extracellular matrix turnover. Together, these findings indicate that DUX4 induces a unique phenotype in MSCs, characterized by impaired differentiation, oxidative stress, partial myogenic reprogramming, and pro-fibrotic activity, contributing to muscle pathology in FSHD. DUX4 FSHD Mesenchymal stem cells differentiation FAP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder currently untreatable. Several hundred thousand people worldwide suffer from FSHD, making it one of the most common inherited degenerative muscle diseases [ 1 ]. FSHD is characterized by progressive weakening of the facial, shoulder girdle, trunk, pelvic girdle muscles, and eventually most skeletal muscles [ 2 ]. Muscle degeneration in FSHD involves inflammation, infiltration of muscle tissue by inflammatory cells [ 3 ], and significant expansion of fibro-adipogenic progenitors [ 4 ]. These processes lead to fibrosis, the replacement of affected muscle tissue with fat and connective tissues [ 5 ]. FSHD is associated with derepression of the D4Z4 macrosatellite repeat of the subtelomeric region of chromosome 4q35 resulting in the aberrant expression of the DUX4 gene [ 6 – 8 ]. DUX4 is a germline transcription factor that is highly expressed during early embryogenesis [ 9 ]. Normally, this expression is suppressed in adult somatic tissues, including human skeletal muscles [ 10 , 11 ]. DUX4 expression is toxic to somatic cells in culture and leads to muscle atrophy in vivo [ 12 , 13 ]. DUX4 expression in human myoblasts induces the production of factors involved in inflammation, such as the chemokine SDF1 (CXCL12) and its receptor CXCR4, which act as chemoattractants for MSCs (mesenchymal stem cells) and inflammatory cells. Following muscle injury, MSCs are released from subcutaneous fat, migrate to the damaged muscle site, and become part of the fibroadipogenic precursor (FAP) compartment. Consequently, the FAP population in regenerating muscle includes both resident FAP and infiltrated fat cells [ 14 – 16 ]. MSCs also actively migrate toward human myoblasts with elevated DUX4 expression [ 17 , 18 ]. This process is blocked by anti-CXCR4 or anti-SDF1 antibodies. Functional assays demonstrated that DUX4-expressing FSHD myoblasts recruit MSCs via the CXCL12-CXCR4 axis to the inflammatory site, where the cells mutually influence each other's proliferation, differentiation, and secretory capacity [ 18 ]. It is noteworthy that most of the results discussed above were obtained on MSСs from healthy subjects; however similarly to myoblasts, MSCs of FSHD patients can also express DUX4 [ 19 ]. We hypothesize that MSCs recruited to sites of injury and involved in tissue repair are also exposed to the pathogenic DUX4 signal. This hypothesis is based on evidence that, in response to inflammation, MSCs form actin- and microtubes-dependent tunneling nanotubes (TNTs) and utilize them for intercellular exchange of content, including organelles, proteins, and mRNA [ 20 – 22 ]. This is possible in the same way for molecular components associated with DUX4 activity. MSCs form TNTs not only among themselves [ 23 ] but also with other cell types, including muscle and muscle-like cells [ 23 – 25 ], DUX4-expressing cells are able to exert a detrimental effect on neighboring DUX4-negative nuclei and cells in mixed muscle structures, demonstrating the possibility of “spreading” the pathological effect beyond the immediate DUX4-expressing myonuclei [ 26 , 27 ]. To assess the effect of DUX4 expression on MSCs in the context of FSHD, we constructed a cellular model of MSCs with inducible ectopic DUX4 expression (MSC-DUX4) and analyzed its gene expression and differentiation potential. Surprisingly, we observed that DUX4 expression induced a reduction of adipogenic and osteogenic potential, disruption in extracellular matrix remodeling and angiogenesis factors, but an increased, but limited capacity for myogenic differentiation. Materials and Methods Cell cultures Human mesenchymal stromal cells (MSCs) derived from adipose tissue were obtained from the Cell Culture Collection of the IDB RAS, Moscow, Russia. They were cultured in DMEM/F12 (Paneco) supplemented with 10% fetal bovine serum (FBS; HyClone), Glutamax (Gibco), penicillin–streptomycin (Gibco), and Insulin-Transferrin-Selenium supplement (ITS; Gibco). Cells are available free of charge upon request from the Cell Culture Collection of the IDB RAS, Moscow, Russia. Immortalized myoblasts derived from a healthy individual (AB1190) and from a FSHD patient (AB1080) [28] were a kind gift of Dr. V. Mouly (Institute of Myology, Paris, France). AB1080 and AB1190 were cultured in a proliferation medium composed of four parts of high-glucose DMEM and one part of Medium 199 (Sigma-Aldrich #M4530) supplemented with 20 % FBS (Life technology #10270), 25 µg/ml Fetuine (Life technology #10344026), 5 ng/ml Human epidermal growth factor (Life technology #PHG0311), 0.5 ng/ml Basic fibroblast growth factor (Life technology #PHG0026), 5 µg/ml Insulin (Sigma #91077C-1G), 0.2 µg/ml Dexamethasone (Sigma #D4902), 1 % penicillin– streptomycin (Gibco #15140-122). Cells are available free of charge upon request from the Institute of Myology, Paris, France. Construction of the MSC-DUX4 cell line We created a model of MSC with inducible DUX4 expression (MSC-DUX4) using the approach described elsewhere [29]. Briefly, the pCW57.1 - DUX4 - WT vector (Addgene plasmid #99282) was transfected into the HEK293T packaging cell line. The viral supernatant was collected 48 and 72 hours post-transfection and filtered. For transduction, MSCs were seeded to form a complete confluent monolayer and then incubated with the lentiviral particles containing the inducible DUX4 construct and a puromycin resistance gene in the presence of polybrene for 24 hours. The following day, the viral-containing medium was replaced with fresh growth medium. After an additional 24-hour incubation in the growth medium, selection was initiated with 0.75 μg/mL puromycin and maintained for 5 days, with medium was changed every 48 hours. Puromycin concentration was determined experimentally for MSCs. Following selection, the cells proliferated and formed dense colonies. DUX4 expression was induced by adding 1 μg/mL doxycycline for 48 hours unless otherwise stated in the text. Differentiation induction Myogenic differentiation Myoblasts were seeded at >95% confluence to form a complete monolayer. After 24 hours, the growth medium was replaced with a differentiation-inducing medium composed of high-glucose DMEM supplemented with 2% horse serum (Paneco), Glutamax, and Penicillin–Streptomycin. No medium changes were performed throughout the differentiation period. On day 5, myotubes were stained with May–Grünwald–Giemsa. MSCs were seeded at >95% confluence to form a complete monolayer. After 24 hours, the growth medium was replaced with a differentiation-inducing medium composed of high-glucose DMEM supplemented with 2% horse serum, Glutamax, Penicillin-Streptomycin, 1 ng/ml bFGF, hydrocortisone (Sigma) and 0,1 µM dexamethasone. Medium changes were performed every three days throughout the differentiation period. On day 10, myotube-like structures were stained with May–Grünwald–Giemsa. Microscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera. For each sample, at least 10 randomly selected fields were imaged, and measurements were analyzed in ImageJ. Myotube fusion index (FI) were quantified, where FI was calculated as the percentage of nuclei located within myotubes relative to the total number of nuclei. Osteogenic differentiation MSCs were seeded at >95% confluence to form a complete monolayer. After 24 hours, the standard growth medium was replaced with an osteogenic induction medium: high-glucose DMEM, 10% FBS, 0,1 µM dexamethasone, 50 µM ascorbate-2-phosphate (Sigma), 10 mM β-glycerophosphate (Sigma). MSCs were cultured for 21 days in the induction medium; the medium was changed every three days during differentiation . Alizarin Red S staining was performed followed by quantitative analysis of mineralization and calcium deposition on day 21 of differentiation. Microscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera. Adipogenic differentiation MSCs were seeded at >95% confluence to form a complete monolayer. After 24 hours, the standard growth medium was replaced with an adipogenic medium: high-glucose DMEM, 10% FBS, 3-Isobutyl-1-methylxanthine (IBMX; Sigma), 1 µM dexamethasone, ITS, 200 µМ indometacin (Sigma). Oil Red O staining was performed followed by quantitative analysis of neutral lipid content on day 21 of differentiation. Microscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera. Immunofluorescence staining Cells were rinsed with PBS (Paneco), fixed in 4% paraformaldehyde (PFA) for 5–10 minutes at 4 °C, and incubated for 30 minutes in a blocking buffer containing 0.1% Triton X-100 (Sigma-Aldrich) and 4% FBS in PBS. Primary antibodies (Table S1) , diluted in the same blocking buffer, were applied overnight at 4 °C in a humid chamber, then secondary antibodies (Table S2) were added for 1 hour at room temperature (RT). Nuclei were counterstained with DAPI (Sigma). Imaging was performed using an Olympus IX73 inverted fluorescence microscope equipped with an Olympus DP camera. For each sample, at least 10 random fields were captured and analyzed using ImageJ as described elsewhere [18]. May-Grunwald Giemsa staining MSCs or myoblasts were stained with May-Grunwald Giemsa dye as previously described [30] after myogenic differentiation. The cells were washed with PBS, fixed with 100% methanol at + 4°C for 5-10 minutes and air-dried. Then the cells were incubated in May-Grunwald's solution (eosin-methylene blue) (1:3 dilution in 1 mM PBS pH 5,6) (MiniMed) for 20 minutes and Giemsa solution (1:20 dilution in 1mM PBS pH 5,6) (Paneco) for 40 minute and washed with distilled water. Alizarin Red S staining After 21 days of osteogenic differentiation, MSCs were washed with PBS and fixed with 4% PFA for 15 minutes at 4°C. Cells were then stained with 40 mM Alizarin Red S (Sigma) for 2 minutes at RT, followed by rinsing with 1 mM HCl in 95% ethanol. Imaging was performed using an Olympus IX73 inverted phase microscope equipped with an Olympus DP camera. For quantitative assessment of mineralization, cells were stained with 40 mM Alizarin Red S (ARS) for 20-30 minutes at RT. Subsequently, 10% acetic acid was added, and the samples were incubated on a shaker at RT for 30 minutes. The acid solution containing stained cells was collected using a cell scraper, transferred to tubes, and heated at 85°C for 10 minutes. After incubation on ice for 5 minutes, the slurry was centrifuged at 20,000 × g for 15 minutes. The supernatant was transferred to a new tube and neutralized with 10% ammonium hydroxide. The neutralized solution was aliquoted in triplicate into a 96-well plate. ARS standards were prepared and aliquoted similarly. Absorbance was measured at 405 nm using a microplate reader (Synergy H1 plate reader, BioTek), and ARS concentration was calculated based on a standard curve generated in Excel. Oil Red O staining After 21 days of adipogenic differentiation, MSCs were washed with PBS and fixed with 4% PFA for 15 minutes at 4°C. Cells were then briefly treated with 60% isopropanol (Macklin) and stained with 0.5% Oil Red O (Sigma-Aldrich) for 10-15 minutes at RT. Following staining, cells were rinsed with 60% isopropanol and washed gently with distilled water. Nuclei were counterstained with Mayer's hematoxylin for 5 minutes. Imaging was performed using an Olympus IX73 inverted microscope equipped with an Olympus DP camera. For quantitative assessment of neutral lipid accumulation, the Oil Red O dye was eluted with 100% isopropanol. The resulting solution was aliquoted in triplicate into 96-well plate, and absorbance was measured at 510 nm using a microplate reader (Synergy H1 plate reader, BioTek). Flow cytometry Cells were detached with 0.05% trypsin–EDTA, washed in PBS by centrifugation at 300 g for 10 minutes, and fixed in Cytofix (BD Biosciences) for 20 minutes at 4 °C. After another wash with PBS, cells were incubated overnight at 4 °C with primary antibodies anti-DUX4 ( Table S2) ; then appropriate secondary antibodies were applied for 1 hour at RT. Samples were washed in PBS again three times aControl samples without primary antibodies were included to assess nonspecific secondary antibody binding. For flow cytometry analysis of MSCs surface markers, cells were detached using 0.05% trypsin-EDTA and washed in PBS (centrifugation at 1300 rpm for 5 minutes). Subsequently, cells were incubated with conjugated antibodies (anti-CD90, anti-CD73, anti-CD44, anti-CD105; Table S3 ) for 1 hour at +4°C, washed three times, and then fixed with Cytofix for 20 minutes at +4°C. Following PBS washes three times (centrifugation at 1600 rpm for 5 min at +4°C) and resuspension in Staining buffer (BD Biosciences). Samples were analyzed on an Attune NxT flow cytometer. MTT test Cells were seeded in 96-well plates (10⁵ cells/well) in the growth medium. After 24 hours MTT (thiazolyl blue tetrazolium bromide; Sigma-Aldrich) was added to the culture medium at a final concentration of 0.5 mg/mL, followed by incubation for 3.5 hours at 37°C. The medium was then carefully removed, and dimethyl sulfoxide (DMSO; Paneco) was added to solubilize the formazan crystals. The plates were incubated for 15 minutes at RT. The resulting solution was aliquoted in triplicate into a 96-well plate, with pure DMSO used as a blank. Absorbance was measured at 590 nm with a reference filter of 620 nm using a microplate reader (Synergy H1 plate reader, BioTek). Absorbance values are proportional to the number of metabolically active cells. Cell viability was expressed as a percentage relative to the control group, and survival curves were generated based on these measurements in Excel. ROS Detection Assay Cells were seeded in 96-well plates (10⁵ cells/well) in the growth medium and allowed to attach overnight. MSCs were washed with PBS. DCFDA Cellular ROS Detection Assay Kit (Abcam, ab113851) was used according to the manufacturer's instructions. Cells were washed with PBS and incubated with 25 μM DCFDA at 37°C in the dark. Fluorescence levels were measured in control MSC and Dox-induced MSC-DUX4 at 30, 60, and 120 minutes after DCFDA addition using a microplate reader (excitation wavelength: 485 nm; emission wavelength: 535 nm; green channel) (Varioskan, Thermo Fisher). Fluorescence intensity was proportional to intracellular ROS levels. Collagen assay Cells were seeded in 6-wells plates (5*10 5 cells) in the growth medium. After 24 hours, the growth medium was replaced with the medium containing 2% FBS and DUX4 expression was induced by adding 1 μg/mL doxycycline. After five days, collagen content was quantified using the Sircol Soluble Collagen Assay Kit (Biocolor). Cultures were washed with PBS and incubated overnight at 4 °C in 0.5 M acetic acid with 0.1 mg/ ml pepsin on a rotating platform. Cells were scraped, centrifuged at 3000 g for 10 minutes at 4 °C, and the medium was collected and cleared by centrifugation at 1500 g for 10 minutes at 4 °C. The Acid Neutralising Reagent was added to the lysates, followed by the addition of cold Isolation & Concentration Reagent to each tube. Samples were incubated overnight at 4°C and then centrifuged at 12,000 × rpm for 10 minutes. The supernatant was removed and Sircol Dye Reagent was added to the pellet. Tubes were capped and mixed by inversion, followed by incubation on a gentle mechanical shaker for 30 minutes to allow formation and precipitation of the collagen-dye complex. After incubation for 30 minutes with gentle shaking, tubes were centrifuged at 12,000 × rpm for 10 minutes. The supernatant was discarded, and the pellet was washed with ice-cold Acid-Salt Wash Reagent to remove unbound dye. Tubes were centrifuged again at 12,000 × rpm for 10 minutes, and the wash reagent was drained. Alkali Reagent was added to dissolve the bound dye, and tubes were vortexed for 5 minutes. The solution was transferred to a 96-well microplate, and absorbance was measured at 555 nm using a microplate reader (Synergy H1 plate reader, BioTek). Absorbance values were measured against a water blank for reagent blanks, standards, and test samples and collagen concentration was calculated based on a standard curve generated in Excel. RT-qPCR Total RNA was extracted using Quick RNA kit (Zymo research). RNA concentration was measured on a BioPhotometer plus spectrophotometer (Eppendorf). RNA samples were stored at -70°C. Purified RNA was reverse transcribed using the MMLV RT kit (Eurogen). cDNA samples were stored at -20°C. cDNA was then mixed with 1 μl primers (10 mkM final) and 2 μl 5Х HS-SYBR Green Master mix (ROX) (Eurogen) in a final volume was 10 μl and analyzed on Light cycler 96 (Roche). Primer sequences are listed in the Table S3. Reactions were performed in triplicates. Relative gene expression level (RQ) was calculated using the 2 − ΔΔ Ct quantification method relative to GAPDH expression. RNAseq The samples were prepared in four biological replicates. The cells were grown as described above and DUX4 expression was induced by adding 1 μg/mL doxycycline. 48h post-induction, the total RNA was extracted using the Nucleospin RNA isolation kit (Macherey–Nagel, 740955) according to the manufacturer's protocol. A total amount of 1 µg RNA per sample was used as input material for the sequencing library preparations. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The library quality was assessed on the Agilent Bioanalyzer 2100 system. Prepared libraries were sequenced on an Illumina HiSeq2000. Raw paired-end sequence reads were mapped to the human genome (GRCh38) using STAR (release 2.7.3a) [31]. Reads were assigned to genes via featureCounts (v 2.0.0) [32]. Differential expression analysis was performed using the DESeq2 R package [33]. Genes with |log2Fold change| > 1 and adjusted p-values( padj) <0.05 were considered significantly differentially expressed. The results of the differential expression analysis are provided in Table S5 . Enrichment analyses were performed against the HALLMARK gene sets of the Molecular Signature Database (https://www.gsea-msigdb.org/gsea/msigdb/) using Gene Set Enrichment Analysis (GSEA). All genes with padj < 0.05 were used. The gene sets used in the venn diagrams and volcano plots were taken from Molecular Signature Database. The geneset IDs in the MSigDB are mentioned in the corresponding figure legends. The code used to generate the results and figures is deposited to GitHub (https://github.com/annaschwager/MSC_DUX4). Statistical analysis Depending on data distribution, either Student’s two-tail t-test or the nonparametric Mann–Whitney test was applied for pairwise comparisons. For multiple comparisons, the Kruskal–Wallis test with FDR correction was used. Analyses were performed in GraphPad Prism 10. Results MSC-DUX4 cells inducibly express DUX4 To model the effect of DUX4 expression on MSCs, we created a cell line with inducible DUX4 expression (MSC-DUX4) as described elsewhere [34] using a lentiviral vector with doxycycline (Dox)-inducible promoter and the puromycin resistance gene (puroR) (see Materials and Methods). DUX4 expression was induced by adding 1 μg/mL doxycycline and confirmed in MSC-DUX4 cells by immunofluorescence staining ( Figure 1A ), flow cytometry ( Figure 1B ) and PCR analysis (Figure 1C) . It should be noted that even without induction, when cultured in commercial serum not certified as tetracycline-free, MSC-DUX4 exhibited low level DUX4 expression due to promoter leakage similarly to MB135-DUX4 cells [35]. This is clearly visible in flow cytometric analysis of DUX4 expression in MSC-DUX4 cells ( Figure 1B ). After confirming the DUX4 construct integration into the MSCs, we performed flow cytometry on Dox-induced MSC-DUX4 to assess the expression of major MSCs markers. Indeed, Dox-induced MSC-DUX4 cells were similar to the control MSCs and did not express HLA-DR. Over 95% of control MSCs were positive for CD73, CD90, and CD105, consistent with international standards for MSCs identification [36]. Likewise, over 95% of Dox-induced MSC-DUX4 were positive for CD90, ~ 93% for CD73, and ~ 90% for CD105. These results indicate that Dox-induced MSC-DUX4 retained positive expression of the core MSCs markers (CD73, CD90, CD105) as well as the additional marker CD44, while maintaining negative expression of HLA-DR, a phenotype identical to that of the control MSCs ( Figure 1C ). Similar to control MSCs, Dox-induced MSC-DUX4 cells were consistently negative for CD56, confirming the absence of a myogenic phenotype ( Figure S1AB ). DUX4 induction in MB135-DUX myoblasts triggers massive apoptosis within 48 hours [34, 35]. We assessed the viability of MSC-DUX4 cells after DUX4 induction using the MTT assay for 8 days after DUX4 induction (Figure 1D) . In contrast to MB135-DUX4 myoblasts which underwent cell death within 48 hours, MSC-DUX4 cells retained viability for at least eight days after DUX4 induction. As elevated DUX4 expression induces oxidative stress in myoblasts [34, 37, 38], we analyzed intracellular reactive oxygen species (ROS) in Dox-induced MSC-DUX4 cells using DCFDA which diffuses into cells and is oxidized to fluorescent DCF in the presence of ROS. We found that after induction of the DUX4 expression, MSCs accumulated significantly more ROS (Figure 1E) . Real-time impedance-based monitoring with the xCelligence system revealed an approximately two-fold reduction in proliferation rate in MSCs upon induction of DUX4 expression ( Figure S1C ). Analysis of gene expression pattern induced in MSC by DUX4 Similarly to myoblasts, MSCs of FSHD patients can also express DUX4 [39], although the effect of DUX4 on MSCs gene expression has never been evaluated. To screen for the MSC-expressed factors that may contribute to muscle impairment in FSHD, we performed RNA-seq in the Dox-induced MSC-DUX4 and the control MSCs ( Figure 2 ). We observed the upregulation (log2Fold >1, padj < 0.05) of 1716 and downregulation (log2Fold < -1, padj < 0.05) of 1356 genes in MSC-DUX4 cells induced for DUX4 expression compared to the control MSC cells (Table S5) . As expected, we detected the upregulation of several known DUX4 targets, e.g. ZSCAN4 and TRIM43 ( Figure 2A, B; Figure S2A ), as well as the downregulation of the PAX7 signature, another common trait of DUX4 -expressing cells [40] ( Figure 2C, D; Figure S2B ). To screen for the biological themes consistently deregulated in MSC by DUX4 expression, we have performed Gene Set Enrichment Analysis (GSEA) against the hallmark gene sets of the Molecular Signatures Database (MSigDB) ( Figure 2E ). The strongest upregulated pathways turned out to be related to the inflammatory response. Among upregulated genes, IL6, TNF, CCL2/5/7/20, CXCL1/6/10/11, IL1A, IL15, belong to a chemotactic and pro-inflammatory secretome indicating an increased capacity of DUX-expressing MSCs to recruit and activate monocytes, neutrophils, T cells and NK cells as well as promote local inflammation. We have also detected a strong downregulation of the genes related to cell division and cell cycle ( Figure 2E ), consistent with a prominent decrease in the doubling time of the Dox-induced MSC-DUX4 cells ( Figure S1C ). To assess the effect of DUX4 expression on the lineage commitment of the MSCs, we have evaluated the expression of the genes involved in the major MSC differentiation pathways, osteogenic, adipogenic, chondrogenic and myogenic. DUX4 expression resulted in the deregulation of the genes involved in all of these differentiation strategies ( Figure 2F ). Notably, we were intrigued by a relatively large number (30) of upregulated genes in the HALLMARK:MYOGENESIS gene set ( Figure 2F ). Those genes encoded for some of the key actors or markers of muscle development, e.g. TNNT1 (4.3 log2fold, 1.5e-70 padj) - a troponin subunit essential for striated muscle contraction, DMD (1.3 log2fold, 3.9e-37 padj) - a crucial muscle protein connecting the cytoskeleton of a muscle fiber to the extracellular matrix, and SVIL (1.6 log2fold, 3.4e-98 padj) - one of the first costameric proteins to assemble during myogenesis ( Figure 2G ). We have also observed the downregulation of some genes related to the negative regulation of smooth muscle cell differentiation ( Figure S2 ). These results suggested that DUX4-expressing MSC might acquire myogenic capacities. DUX4 confers a limited myogenic potential to mesenchymal stem cells To experimentally test our prediction that DUX4 expression conferred a myogenic potential to MSC-DUX4 cells, we induced myogenic differentiation in the control MSCs and Dox-induced MSC-DUX4 as described elsewhere [18]. In contrast to the control MSCs, Dox-induced MSC-DUX4 cells formed myotube-like structures on day 10 of differentiation whereas normal human myoblasts show this pattern at days 4-6 [18, 35] (Figure S3A) . Myotube-like structures formed by MSC-DUX4 cells were positive for desmin, MF20 (anti-MYH), Troponin T and myogenin. MF20, myogenin, Troponin T are markers of mature myotubes, desmin is a marker for both committed and differentiating myoblasts. We observed that, in contrast to control MSC, Dox-induced MSC-DUX4 cells formed myotube-like structures and expressed key myogenic markers. Immunostaining was positive for MF20, troponin T (markers of late myogenesis; Figure 3A ), and myogenin (a marker of mid myogenesis; Figure S3B ). Furthermore, Dox-induced MSC-DUX4 cells expressed desmin, another early marker of myogenic commitment ( Figure S3B ). Control MSC also exhibited a positive staining for desmin, consistent with its role as an early differentiation marker and its known expression in committed myogenic progenitors. It should be noted that neither control MSCs nor MSC-DUX4 were positive for CD56, a marker of myogenic progenitor cells ( Figure S1AB ). To quantify the efficiency of myotube formation, we calculated the fusion index (FI) defined as the number of nuclei inside the myotubes divided by the total number of nuclei * 100%. The FI of Dox-induced MSC-DUX4 cells was approximately 10-fold lower than that of the control myoblasts, even though the Dox-induced MSC-DUX4 cells were capable of generating myotube-like structures contrary to the control MSCs (Figure 3B) . We next directly compared the expression of myogenic markers (myogenin, desmin and troponin T) in Dox-induced MSC-DUX4 to myoblasts on day 5 of differentiation using quantitative real-time PCR (RT-qPCR).The expression levels of myogenesis markers in Dox-induced MSC-DUX4 were significantly lower or entirely absent compared to even FSHD myotubes (Figure 3C) , suggesting that Dox-induced MSC-DUX4 derived myotubes might be functionally defective. Expression levels of Troponin T2 were 0.00002 ± 0.0 vs. 0.00006 ± 0. 00001 vs. 1.30134 ± 0.06505 for MSCs, Dox-induced MSC-DUX4 and FSHDmyoblasts respectively; expression levels of myogenin were: 0 vs. 0 vs. 0.13584 ± 0.00942 for MSCs and Dox-induced MSC-DUX4 and FSHD-myoblasts respectively; expression levels of desmin: 0 vs 0.00035 ± 0.00003 vs 1.85318 ± 0.12845 for MSCs and Dox-induced MSC-DUX4 and FSHD-myoblasts, respectively. We thus made an exciting discovery that Dox-induced MSC-DUX4 were to some extent capable of forming myotubes, in contrast to the control MSC. However, these structures may not represent mature, functional myotubes comparable to those derived from bona fide myoblasts. DUX4 reduces the capacity of MSC for the adipogenic and osteogenic differentiation Next, we compared differentiation capacities of the control MSC and Dox-induced MSC-DUX4 for the adipogenic and osteogenic differentiation. MSCs were cultured for 21 days in either osteogenic or adipogenic induction medium as described in the Material and Methods. Osteogenic differentiation was evaluated by Alizarin Red S staining followed by quantitative analysis of extracellular matrix mineralization (calcium deposition) as described in Material and Methods (Figure 4A) . Adipogenic differentiation was evaluated by Oil Red O staining followed by quantitative analysis of neutral lipid content as described in Material and Methods (Figure 4B) . In contrast to the control MSC, Dox-induced MSC-DUX4 cells had a reduced capacity for osteogenic and adipogenic differentiation. Dox-induced MSC-DUX4 accumulated approximately four-fold less lipids (0.16 ± 0.01 vs. 0.68 ± 0.03 OD 510 ) compared to the control MSC ( Figure 4A ) and approximately two times less calcium 3.54 ± 0.56 vs.(7.3 ± 1.7 mM) ( Figure 4B ). The expression of an early adipogenic marker FABP4 and a late marker leptin was analyzed by immunostaining ( Figure S4A ) and RT-qPCR ( Figure S4B ) on the 5th, 15th and 21th day of differentiation. FABP4 was expressed at high levels on day 5 of differentiation in the control MSCs. Its expression then decreased approximately two-fold and remained stable until day 21. Leptin expression in the control MSCs exhibited a wave-like pattern: it increased by day 5, declined to undetectable levels by day 15, and increased again by day 21. In contrast, Dox-induced MSC-DUX4 showed markedly altered expression profiles. First, the overall expression level of FABP4 was two times lower compared to the controls. The dynamics of FABP4 expression was also different: instead of peaking on day 5, expression continued to increase until day 15, indicating a clear temporal shift in the differentiation. Leptin expression was nearly absent in Dox-induced MSC-DUX4, suggesting a failure to undergo terminal adipogenic differentiation. Together, these results indicate that high levels of DUX4 expression impair the adipogenic differentiation potential of MSCs. DUX4 induces pro-fibrotic activity of MSCs We have previously demonstrated that MSCs could produce and secrete extracellular matrix proteins, such as collagen and fibronectin, thereby contributing to fibrosis development in the context of FSHD [18]. Here we assessed the contribution of DUX4-expressing MSC to the extracellular matrix (ECM) production. We have observed transcriptional upregulation of 45 genes comprising the matrisome in the Dox-induced MSC-DUX4 cells, including 8 collagen-encoding genes and 35 genes encoding structural ECM glycoproteins ( Figure 5AB ). We have thus experimentally evaluated the collagen synthesis and secretion of collagen, a major ECM protein in the DUX4-expressing and control MSC. Control MSC and Dox-induced MSC-DUX4 were immunocytochemically stained for collagen types III and IV ( Figure 5C ). Secreted collagen appears as thin filaments in the extracellular space(yellow arrows in Figure 5C ). We found that Dox-induced MSC-DUX4 exhibited an increased level of collagen types III and IV secretion compared to the control MSC. Next, we quantified collagen accumulation in lysates of the control and Dox-induced MSC-DUX4 using Sircol Soluble Collagen Assay Kit . Collagen concentration in lysates was measured five days post-DUX4 induction. We detected a two-fold increase in collagen content in Dox-induced MSC-DUX4 lysates compared to the control MSC (Figure 5D) . Additionally, we stained the control and Dox-induced MSC-DUX4 with antibodies against an ECM glycoprotein fibronectin and observed that Dox-induced MSC-DUX4 secreted thicker fibronectin fibers compared to the control (Figure 5E). Discussion Facioscapulohumeral muscular dystrophy (FSHD) is characterized both by skeletal muscle degeneration and progressive replacement of muscle tissue with fibrotic and adipose tissue [ 42 ]. The skeletal muscle niche harbors a heterogeneous population of mesenchymal stem cells (MSCs), including both resident MSCs and those migrating to the injured site from other adjacent tissues following muscle injury [ 17 , 20 ]; they become part of the fibroadipogenic precursor (FAP) compartment. Consequently, the FAP population in regenerating muscle includes both resident FAP and infiltrated cells [ 14 – 16 ]. In this study, we specifically concentrate on cells migrating into muscle, particularly from adipose tissue. They rapidly infiltrate damaged muscle and significantly contribute to tissue regeneration and remodeling. Recent studies indicate that these mobilized MSCs affect myogenesis [ 15 , 38 , 39 ]. Since contraction of the 4q35 D4Z4 array or its derepression leading to DUX4 expression is a hallmark of FSHD [ 6 ], reviewed in [ 7 ], we hypothesized that MSCs recruited to sites of injury and involved in tissue repair are also exposed to the pathogenic DUX4 signal. This hypothesis is based on evidence that, in response to inflammation, MSCs form actin- and microtubule-dependent tunneling nanotubes (TNTs) and utilize them for intercellular exchange of content, including organelles, proteins, and mRNA [ 21 – 23 ]. This is possible in the same way for molecular components associated with DUX4 activity. MSCs form TNTs not only among themselves [ 24 ] but also with other cell types, including muscle and muscle-like cells [ 24 – 26 ], making TNT-mediated communication with myoblasts highly plausible and creating a route for the intercellular transfer of molecular components associated with DUX4 activity. In addition, DUX4-expressing cells are able to exert a detrimental effect on neighboring DUX4-negative nuclei and cells in mixed muscle structures, demonstrating the possibility of “spreading” the pathological effect beyond the immediate DUX4-expressing myonuclei [ 27 , 43 ]. Together, these observations support the development of the MSC-DUX4 cellular model to investigate the contribution of DUX4 expression in non-myoblasts cell types involved in muscle regeneration and their intercellular communication in the pathogenesis of FSHD. Following doxycycline induction, MSC-DUX4 cells robustly expressed DUX4 protein while retaining their characteristic MSC phenotype, including positive expression of CD73, CD90, and CD105, and the absence of HLA-DR and CD56. This stability in surface marker expression confirms that DUX4 induction does not compromise mesenchymal identity. However, functional assays revealed a pronounced reduction in proliferation rate and a marked accumulation of reactive oxygen species (ROS), indicating that DUX4 imposes cellular stress in MSCs similar to that seen in myoblasts [ 37 , 38 , 44 ] This validates our system as a suitable model for dissecting DUX4-related mechanisms in a mesenchymal context. An important difference emerged when comparing the survival of MSC-DUX4 to previously reported myoblast models ectopically expressing DUX4. While myoblasts rapidly undergo apoptosis upon DUX4 activation [ 34 , 37 , 38 ], MSC-DUX4 cells remain viable for at least eight days post-induction, suggesting intrinsic differences in stress tolerance and survival mechanisms between these cell types. This differential sensitivity may reflect the greater adaptability of MSCs to oxidative and metabolic stress, consistent with their role as stromal support cells in variable tissue environments [ 45 , 46 ]. Transcriptomic analysis revealed extensive gene deregulation upon DUX4 induction, with more than 1,700 upregulated and 1,300 downregulated transcripts. The upregulation of canonical DUX4 targets, such as ZSCAN4 and TRIM43 , validates the functionality of the system. Conversely, downregulation of PAX7 target genes previously identified as a hallmark of DUX4 activity [ 40 ] was also evident, confirming that the DUX4 transcriptional network was faithfully recapitulated in MSCs. Gene set enrichment analysis (GSEA) revealed strong induction of inflammatory and immune response pathways, including the upregulation of IL6 , TNF , CCL2/5/7/20 , and CXCL1/6/10/11 , all components of a potent pro-inflammatory secretome. These changes imply that DUX4-expressing MSCs could serve as active mediators of local inflammation and immune cell recruitment in FSHD muscle, an emerging theme in recent studies of disease progression. Another finding was the significant transcriptional suppression of genes related to cell cycle and proliferation, in agreement with the observed reduction in doubling time. This suggests that while DUX4-expressing MSCs remain viable, their proliferative capacity is impaired, potentially limiting their regenerative contributions within muscle tissue. Interestingly, DUX4 also disrupted lineage-specific gene programs, simultaneously affecting osteogenic, adipogenic, chondrogenic, and myogenic differentiation pathways. Particularly noteworthy was the enrichment of the HALLMARK:MYOGENESIS gene set, including upregulation of TNNT1 , DMD , and SVIL , as well as the downregulation of negative regulators of myogenesis. These transcriptional changes predicted an unexpected partial acquisition of myogenic traits by MSC-DUX4. Indeed, functional differentiation assays confirmed that DUX4 expression conferred limited myogenic capacity to MSCs. Dox-induced MSC-DUX4 cells formed myotube-like structures expressing desmin, myogenin, troponin T, and MYH, markers characteristic of various stages of myogenesis. However, these myotube-like structures were sparse and morphologically immature, and their fusion index was approximately ten times lower than that of normal myoblasts. Quantitative PCR further revealed low or absent expression of myogenic markers compared even with FSHD myotubes, suggesting that while DUX4 can initiate partial myogenic reprogramming, it cannot sustain a complete or functional myogenic differentiation program in MSCs. This partial reprogramming may reflect a transient activation of myogenic transcriptional modules by DUX4, insufficient for terminal differentiation. In parallel, DUX4 impaired the canonical differentiation potential of MSCs. Both osteogenesis and adipogenesis were markedly reduced, with disrupted temporal dynamics of adipogenic markers (FABP4, leptin). These findings suggest that DUX4 reprograms MSCs fate, diverting them away from their physiological lineages and potentially altering tissue homeostasis. Conversely, reductions in whole-body and regional bone mineral density (BMD) are associated with diminished muscle strength and functional capacity in FSHD patients who also show an increased frequency of traumatic fractures [ 47 ]. One of the most striking effects of DUX4 induction was the enhancement of pro-fibrotic activity. DUX4-expressing MSCs showed transcriptional upregulation of numerous matrisome genes, including those encoding collagens and structural ECM components. Immunocytochemical and biochemical assays confirmed elevated synthesis and secretion of collagen types III and IV, as well as thickened fibronectin fibers. Given the established role of MSCs in fibrosis in the FSHD context [ 18 ], this effect provides a compelling mechanism by which DUX4 expression in MSCs could exacerbate the fibrotic environment characteristic of FSHD muscle [ 48 ].This is consistent with the described upregulation of fibroadipogenic progenitor gene expression in samples from FSHD patients and model organisms [ 5 , 49 ]. Taken together, our findings redefine the role of DUX4 in FSHD pathology by demonstrating that MSCs, in addition to myoblasts, may represent an additional target of DUX4-mediated reprogramming. In MSCs, DUX4 promotes oxidative stress, inflammation, and fibrosis while impairing normal differentiation and regeneration. The partial acquisition of myogenic traits coupled with a pro-fibrotic secretory profile suggests that MSC-DUX4 may contribute to the aberrant tissue remodeling characteristic of FSHD. However in our system, the apparent concurrent induction of myogenic and profibrotic programs likely reflects cell‑to‑cell heterogeneity of DUX4 levels rather than their co‑activation in the same cells. These processes are spatially segregated across different subpopulations. Endogenous DUX4 expression in FSHD myogenic cells is well known to be rare, stochastic and heterogeneous, with only a small fraction of nuclei/cells showing a high DUX4/DUX4‑target signature at any given time [ 50 – 52 ]. RNA-seq and MERFISH show the separation of “FSHD-hghi” and “FSHD-low” clusters/states by DUX4 targets, i.e., transcriptional heterogeneity within a single culture and even a single myotube, and the FSHD profile is determined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses [ 53 – 55 ]. A similar pattern is observed in our MSC‑DUX4 model (Fig. 1 ). At high DUX4 levels, apoptosis and severe differentiation impairments predominate [ 56 , 57 ]. At lower DUX4 levels, myogenesis is impaired without an immediate cell death [ 58 , 59 ]. Even \"transient DUX4 bursts\" leave a persistent profibrotic state of FAP-like cells and an increased susceptibility to fibrosis [ 56 ]. Therefore, we propose that MSCs with low or absent expression follow the canonical adipogenic trajectory for MSCs, while DUX4-positive cells form defective myotubes and/or activate profibrotic transcriptional programs. Future work should aim to determine whether these DUX4-induced alterations in MSC behavior influence muscle repair and intercellular communication in vivo, and whether targeting DUX4 signaling in stromal cells could mitigate fibrotic progression and improve muscle function in FSHD. Abbreviations ARS: Alizarin Red S BCA: Bicinchoninic Acid Assay BMD: Bone Mineral Density C2C12: Mouse Myoblast Cell Line CCL2: C-C Motif Chemokine Ligand 2 CD: Cluster of Differentiation CXCL1: C-X-C Motif Chemokine Ligand 1 CXCL12: C-X-C Motif Chemokine Ligand 12 CXCR4: C-X-C Chemokine Receptor 4 D4Z4: D4Z4 Macrosatellite Repeat on Chromosome 4 DAPI: 4′,6-Diamidino-2-Phenylindole DCF: Dichlorofluorescein DCFDA: 2′,7′-Dichlorodihydrofluorescein Diacetate DMEM: Dulbecco’s Modified Eagle Medium DMSO: Dimethyl Sulfoxide DNA: Deoxyribonucleic Acid DPBS: Dulbecco’s Phosphate-Buffered Saline DUX4: Double Homeobox 4 ECL: Enhanced Chemiluminescence ECM: Extracellular Matrix EDTA: Ethylenediaminetetraacetic Acid FABP4: Fatty Acid Binding Protein 4 FAP: Fibro-Adipogenic Progenitor FBS: Fetal Bovine Serum FDR: False Discovery Rate FSHD: Facioscapulohumeral Muscular Dystrophy FSHD1: Facioscapulohumeral Muscular Dystrophy Type 1 FSHD2: Facioscapulohumeral Muscular Dystrophy Type 2 GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase GSEA: Gene Set Enrichment Analysis H3K9: Histone H3 Lysine 9 HEK293T: Human Embryonic Kidney 293T Cells HLA-DR: Human Leukocyte Antigen DR IBMX: 3-Isobutyl-1-Methylxanthine IL: Interleukin ITS: Insulin-Transferrin-Selenium Supplement MERFISH: Multiplexed Error-Robust Fluorescence In Situ Hybridization MMLV: Moloney Murine Leukemia Virus MRI: Magnetic Resonance Imaging MSC: Mesenchymal Stromal Cell MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide MYH: Myosin Heavy Chain NMD: Nonsense-Mediated mRNA Decay PBS: Phosphate-Buffered Saline PBST: Phosphate-Buffered Saline with Tween-20 PCR: Polymerase Chain Reaction PFA: Paraformaldehyde PAX7: Paired Box Protein 7 ROS: Reactive Oxygen Species Declarations Ethics approval and consent to participate Consent for publication Availability of data and materials: Data and material will be made available on reasonable request. Competing interests The authors declare that they have no competing interests Funding This study was supported by FSHD Society (USA) and the IDB RAS Government basic research program (0088-2024-0010) to YV and the Russian Science Foundation Grant No. 25-15-00443 to ED. Authors' contributions YV and EK devised the experiments; OS, AS, EK and ED carried out the experiments; OS, AS, EK, EV and YV analyzed data, OS, AS, EK and YV wrote the main manuscript text and prepared figures; YV and ED obtained funding. All authors reviewed and approved the manuscript. Data availability All relevant data are within the paper and the Supplementary Data. The code used to generate the results and figures is deposited to GitHub (https://github.com/annaschwager/MSC_DUX4).. References Mostacciuolo M, Pastorello E, Vazza G, Miorin M, Angelini C, Tomelleri G, Galluzzi G, Trevisan C (2009) Facioscapulohumeral muscular dystrophy: epidemiological and molecular study in a north‐east Italian population sample. 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Epigenetics Chromatin 11:47. https://doi.org/10.1186/s13072-018-0215-z Rickard AM, Petek LM, Miller DG (2015) Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways. Hum Mol Genet 24:5901–5914. https://doi.org/10.1093/hmg/ddv315 Jiang S, Williams K, Kong X, Zeng W, Nguyen NV, Ma X, Tawil R, Yokomori K, Mortazavi A (2020) Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. PLOS Genet 16:e1008754. https://doi.org/10.1371/journal.pgen.1008754 Zheng D, Wondergem A, Kloet S, Willemsen I, Balog J, Tapscott SJ, Mahfouz A, Van Den Heuvel A, Van Der Maarel SM (2024) snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis. Hum Mol Genet 33:284–298. https://doi.org/10.1093/hmg/ddad186 Williams K, Kong X, Zeng W, Nguyen NV, Ma X, Tawil R, Yokomori K, Mortazavi A (2020) Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. PLOS Genet 16:e1008754. https://doi.org/10.1371/journal.pgen.1008754 Chen L, Kong X, Johnston KG, Mortazavi A, Holmes TC, Tan Z, Yokomori K, Xu X (2024) Single-cell spatial transcriptomics reveals a dystrophic trajectory following a developmental bifurcation of myoblast cell fates in facioscapulohumeral muscular dystrophy. Genome Res 34:665–679. https://doi.org/10.1101/gr.278717.123 Bosnakovski D, Oyler D, Mitanoska A, Douglas M, Ener ET, Shams AS, Kyba M (2022) Persistent Fibroadipogenic Progenitor Expansion Following Transient DUX4 Expression Provokes a Profibrotic State in a Mouse Model for FSHD. Int J Mol Sci 23:1983. https://doi.org/10.3390/ijms23041983 Knopp P, Krom YD, Banerji CRS, Panamarova M, Moyle LA, den Hamer B, van der Maarel SM, Zammit PS (2016) DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis. J Cell Sci 129:3816–3831. https://doi.org/10.1242/jcs.180372 Bosnakovski D, Xu Z, Ji Gang E, Galindo CL, Liu M, Simsek T, Garner HR, Agha‐Mohammadi S, Tassin A, Coppée F, Belayew A, Perlingeiro RR, Kyba M (2008) An isogenetic myoblast expression screen identifies DUX4‐mediated FSHD‐associated molecular pathologies. EMBO J 27:2766–2779. https://doi.org/10.1038/emboj.2008.201 Bosnakovski D, Gearhart MD, Toso EA, Ener ET, Choi SH, Kyba M (2018) Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression. Sci Rep 8:16957. https://doi.org/10.1038/s41598-018-35150-8 Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.docx Supplementarydata.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9094205\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":609454399,\"identity\":\"a09e6d74-1e63-4939-a865-5ae185fb75db\",\"order_by\":0,\"name\":\"Olesya SERBINA\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Koltzov Institute of Developmental Biology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Olesya\",\"middleName\":\"\",\"lastName\":\"SERBINA\",\"suffix\":\"\"},{\"id\":609454400,\"identity\":\"75f67249-75cf-4fda-8910-01c6e9174010\",\"order_by\":1,\"name\":\"Anna SCHWAGER\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institut Gustave Roussy\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Anna\",\"middleName\":\"\",\"lastName\":\"SCHWAGER\",\"suffix\":\"\"},{\"id\":609454401,\"identity\":\"7a32069e-d7ef-4198-9713-b7b3c4ade7a4\",\"order_by\":2,\"name\":\"Ekaterina KISELEVA\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Koltzov Institute of Developmental Biology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ekaterina\",\"middleName\":\"\",\"lastName\":\"KISELEVA\",\"suffix\":\"\"},{\"id\":609454402,\"identity\":\"c3cb8243-8caf-498b-9008-06865cb07873\",\"order_by\":3,\"name\":\"Erdem DASHINIMAEV\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Koltzov Institute of Developmental Biology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Erdem\",\"middleName\":\"\",\"lastName\":\"DASHINIMAEV\",\"suffix\":\"\"},{\"id\":609454403,\"identity\":\"41e1fe1e-2597-4f1c-bfa6-2db6afa26c1a\",\"order_by\":4,\"name\":\"Ekaterina VOROTELYAK\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Koltzov Institute of Developmental Biology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ekaterina\",\"middleName\":\"\",\"lastName\":\"VOROTELYAK\",\"suffix\":\"\"},{\"id\":609454404,\"identity\":\"fa33dcea-b48e-48b5-89ce-0bbbda0be4bf\",\"order_by\":5,\"name\":\"Yegor Vassetzky\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYPCDAwxyEIYNceoZG4BajCHsNBK0JDYQ0mJwvP3i4wqGw/Ly/YePP/xx5nB6/+zexx8YEu7h1nLmTLHhGYbDhhtupCU2SNw4nDvjznEzCYaEYtxabuSkSTYwpDFukOAxbDD4cDh3g0QaGwPjjwTcWu6/AWuxn99//mNDwofD6QYSacxAh+HRcoP9GFCLTWLDgRzGhgM3DicAtTBI4NMieSaHGeggm2SgXwxnNpxJN5xxI41NIgGPFr7jxx8+bKiQsJ3ff/jBxx/HrOX5ZwAd9gGPFoUDPAZA58H5zRAKtwYGBvkG9gfI/Do8akfBKBgFo2CkAgAyjVzYoyRvZAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Institut Gustave Roussy\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yegor\",\"middleName\":\"\",\"lastName\":\"Vassetzky\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-11 12:38:56\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9094205/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9094205/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":105198490,\"identity\":\"13972e08-1674-46eb-b9d6-de7f73165d7a\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:35\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":278145,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDUX4 expression in MSCs. A, \\u003c/strong\\u003eRepresentative images of immunohistochemical staining for DUX4 (green) in the control MSC or Dox-induced MSC-DUX4; Nuclei were stained with DAPI; \\u003cstrong\\u003eB,\\u003c/strong\\u003e Flow cytometry data analysis of DUX4 expression; \\u003cstrong\\u003eC, \\u003c/strong\\u003eRelative expression levels of \\u003cem\\u003eDUX4 \\u003c/em\\u003ein the control MSC or Dox-induced MSC-DUX4 calculated according to the 2−∆∆Ct quantification method vs. \\u003cem\\u003eGAPDH\\u003c/em\\u003e(results are presented as mean ± SD; NS, non-significant; * p\\u0026lt;0.05; n = 3); \\u003cstrong\\u003eD\\u003c/strong\\u003e, Flow cytometry data analysis of MSCs surface markers expression (mean ± SD, NS, non-significant; * p \\u0026lt;0.05, n = 3); \\u003cstrong\\u003eE,\\u003c/strong\\u003e MSC-DUX4 survival during 8 days after DUX4 induction, MTT assay data, (mean ± SD, NS, non-significant; * p \\u0026lt;0.05, n = 24); \\u003cstrong\\u003eF,\\u003c/strong\\u003e ROS accumulation in the control MSC and in Dox-induced MSC-DUX4 measured by DCF staining (mean ± SD, NS, non-significant; * p \\u0026lt;0.05, n = 11); Scale bar=50 µm\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/0d147e8eaf382cf1b38d46d1.png\"},{\"id\":105198492,\"identity\":\"f960130d-668d-4b49-805e-cc22bcc809ba\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:35\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":938414,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffect of ectopic \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eDUX4\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e expression on gene expression in MSCs. A\\u003c/strong\\u003e, Venn diagram showing the intersection between the genes significantly upregulated in the Dox-induced MSC-DUX4 vs control MSC (log2Fold \\u0026gt; 1, padj \\u0026lt; 0.05) and the DUX4 signature genes published in \\u003ca href=\\\"https://www.zotero.org/google-docs/?sxiT0j\\\"\\u003e[41]\\u003c/a\\u003e; \\u003cstrong\\u003eB\\u003c/strong\\u003e, Expression of two example DUX4 target genes in the control MSC (red) or Dox-induced MSC-DUX4 (blue); \\u003cstrong\\u003eC\\u003c/strong\\u003e, Venn diagram showing the intersection between the genes significantly downregulated in the Dox-induced MSC-DUX4 vs control MSC (log2Fold \\u0026lt; -1, padj \\u0026lt; 0.05) and the PAX7 target genes (MSigDB:M30110); \\u003cstrong\\u003eD\\u003c/strong\\u003e, Expression of two example PAX7 target genes in the control MSC (red) or Dox-induced MSC-DUX4 (blue); \\u003cstrong\\u003eE\\u003c/strong\\u003e, Gene Set Enrichment Analysis against the HALLMARK genesets of the MSigDB for the genes significantly deregulated in the Dox-induced MSC-DUX4 vs control MSC; \\u003cstrong\\u003eF\\u003c/strong\\u003e, Deregulation of the genes of the four major MSC differentiation programs in the Dox-induced MSC-DUX4. Venn diagrams show the intersection between the genes upregulated (left) or downregulated (right) in the Dox-induced MSC-DUX4 vs control MSC and the corresponding genesets of the MSigDB: GOBP_BONE_CELL_DEVELOPMENT (M11179), HALLMARK_ADIPOGENESIS (M5905), GOBP_CHONDROCYTE_DEVELOPMENT (M11483), HALLMARK_MYOGENESIS (M5909). \\u003cstrong\\u003eG\\u003c/strong\\u003e, Volcano plot showing the genes deregulated in the Dox-induced MSC-DUX4 vs control MSC comparison. Highlighted in red are the upregulated genes (log2Fold \\u0026gt; 1, padj \\u0026lt; 0.05) belonging to the HALLMARK_MYOGENESIS geneset. Highlighted blue are the downregulated genes belonging to the GOBP:NEGATIVE_REGULATION_OF_SMOOTH_MUSCLE_DIFFERENTIATION gene set.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/369bff0e72e7703098823ee9.png\"},{\"id\":105198491,\"identity\":\"c3ce6bd3-3604-47b5-b6cd-64b69d58a22a\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:35\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":922773,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSCs with induced DUX4 expression have a limited potential for myogenic differentiation. A,\\u003c/strong\\u003e Representative images of immunohistochemical staining of the the control MSC and Dox-induced MSC-DUX4 cells after 10 days of myogenic differentiation for MF20 (red) and Troponin T (green); Nuclei are stained with DAPI; \\u003cstrong\\u003eB,\\u003c/strong\\u003e Fusion index (FI) of Dox-induced MSC-DUX4, normal and FSHD myoblasts (results are presented as mean ± SD; NS, non-significant; * p\\u0026lt;0.05; n = 10); \\u003cstrong\\u003eC\\u003c/strong\\u003e, Expression level of myogenic markers in MSC and FSHD myoblasts after 5 days of myogenic differentiation calculated according to the 2−∆∆Ct quantification method vs. \\u003cem\\u003eGAPDH\\u003c/em\\u003e(results are presented as mean ± SD; NS, non-significant; * p\\u0026lt;0.05; n = 3); Scale bar = 200 µm\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/5edac084d343e333efa477a3.png\"},{\"id\":105198494,\"identity\":\"1354ba45-6e96-4121-8578-4d11c6d2227d\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:36\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1841135,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDUX4 expression reduces the capacity of MSCs for the adipogenic and osteogenic differentiation. A, \\u003c/strong\\u003eLeft panel, representative images of Oil Red O staining of MSCs after 21 days of adipogenic differentiation; right panel, absorption of Oil Red O dye eluted from stained MSCs (results are presented as mean ± SD; NS, non-significant; * p\\u0026lt;0.05; n = 6);\\u003cstrong\\u003e \\u003c/strong\\u003eScale bar = 100 µm;\\u003cstrong\\u003e B, \\u003c/strong\\u003eLeft panel, representative images of Alizarin Red S staining of MSCs after 21 days of osteogenic differentiation; right panel, concentration of Alizarin Red S dye eluted from stained MSCs (results are presented as mean ± SD; NS, non-significant; * p\\u0026lt;0.05; n = 6); Scale bar = 200 µm\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/8e096171c5c44f403b1cd67d.png\"},{\"id\":105198495,\"identity\":\"0278c227-31d2-4760-a540-d30577c05390\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:36\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1890354,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSC-DUX4 cells produce ECM. A, \\u003c/strong\\u003eVenn diagram showing the intersection between the genes significantly deregulated in the Dox-induced MSC-DUX4 vs control MSC and the genes encoding the core matrisome proteins (MSigDB: NABA_CORE_MATRISOME (M5884)). \\u003cstrong\\u003eB,\\u003c/strong\\u003e Volcano plot showing the genes deregulated in the Dox-induced MSC-DUX4 vs control MSC comparison. Highlighted in pink are significantly deregulated genes (abs(log2Fold) \\u0026gt; 1, padj \\u0026lt; 0.05) belonging to the MSigDB NABA_ECM_GLYCOPROTEINS geneset (M3008). Highlighted violet are significantly deregulated genes (abs(log2Fold) \\u0026gt; 1, padj \\u0026lt; 0.05) belonging to the MSigDB NABA_COLLAGENS (M3005) geneset. \\u003cstrong\\u003eC\\u003c/strong\\u003e, Representative images of immunohistochemical staining of the control MSC and Dox-induced MSC-DUX4 for collagen type III (top) and collagen type IV (bottom) in the control and TGFβ-treated MSCs; Nuclei are stained with DAPI; \\u003cstrong\\u003eD\\u003c/strong\\u003e, Collagen concentration in the lysates of the control MSC and Dox-induced MSC-DUX4; \\u003cstrong\\u003eE\\u003c/strong\\u003e, Representative images of immunohistochemical staining of the control MSC and Dox-induced MSC-DUX4 for fibronectin in the control and TGFβ-treated MSCs; Nuclei are stained with DAPI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/7d603cdc718b07a75e1c079b.png\"},{\"id\":107481428,\"identity\":\"65859001-971d-4068-967d-7e0de57a0f0a\",\"added_by\":\"auto\",\"created_at\":\"2026-04-22 02:17:54\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":6576757,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/082a15ab-ff28-4e6c-b14c-5355405dd2ca.pdf\"},{\"id\":105198493,\"identity\":\"e5b3fe51-5d86-429e-b42c-8f846db79e09\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:36\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":133172,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Graphicalabstract.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/a9de0a5c79a357403e1c1316.docx\"},{\"id\":105198497,\"identity\":\"00c4de2f-f677-43db-976c-1b458061bd8b\",\"added_by\":\"auto\",\"created_at\":\"2026-03-23 10:45:36\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3851285,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarydata.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9094205/v1/b9de3f3f36013807bab790c6.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Beyond Myoblasts: DUX4 Drives Fibrosis and Myogenic Reprogramming in Mesenchymal Stem Cells\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eFacioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder currently untreatable. Several hundred thousand people worldwide suffer from FSHD, making it one of the most common inherited degenerative muscle diseases [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. FSHD is characterized by progressive weakening of the facial, shoulder girdle, trunk, pelvic girdle muscles, and eventually most skeletal muscles [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Muscle degeneration in FSHD involves inflammation, infiltration of muscle tissue by inflammatory cells [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e], and significant expansion of fibro-adipogenic progenitors [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. These processes lead to fibrosis, the replacement of affected muscle tissue with fat and connective tissues [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. FSHD is associated with derepression of the D4Z4 macrosatellite repeat of the subtelomeric region of chromosome 4q35 resulting in the aberrant expression of the \\u003cem\\u003eDUX4\\u003c/em\\u003e gene [\\u003cspan additionalcitationids=\\\"CR7\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. \\u003cem\\u003eDUX4\\u003c/em\\u003e is a germline transcription factor that is highly expressed during early embryogenesis [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Normally, this expression is suppressed in adult somatic tissues, including human skeletal muscles [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. \\u003cem\\u003eDUX4\\u003c/em\\u003e expression is toxic to somatic cells in culture and leads to muscle atrophy \\u003cem\\u003ein vivo\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. \\u003cem\\u003eDUX4\\u003c/em\\u003e expression in human myoblasts induces the production of factors involved in inflammation, such as the chemokine SDF1 (CXCL12) and its receptor CXCR4, which act as chemoattractants for MSCs (mesenchymal stem cells) and inflammatory cells. Following muscle injury, MSCs are released from subcutaneous fat, migrate to the damaged muscle site, and become part of the fibroadipogenic precursor (FAP) compartment. Consequently, the FAP population in regenerating muscle includes both resident FAP and infiltrated fat cells [\\u003cspan additionalcitationids=\\\"CR15\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. MSCs also actively migrate toward human myoblasts with elevated \\u003cem\\u003eDUX4\\u003c/em\\u003e expression [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. This process is blocked by anti-CXCR4 or anti-SDF1 antibodies. Functional assays demonstrated that DUX4-expressing FSHD myoblasts recruit MSCs \\u003cem\\u003evia\\u003c/em\\u003e the CXCL12-CXCR4 axis to the inflammatory site, where the cells mutually influence each other's proliferation, differentiation, and secretory capacity [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. It is noteworthy that most of the results discussed above were obtained on MSСs from healthy subjects; however similarly to myoblasts, MSCs of FSHD patients can also express \\u003cem\\u003eDUX4\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. We hypothesize that MSCs recruited to sites of injury and involved in tissue repair are also exposed to the pathogenic DUX4 signal. This hypothesis is based on evidence that, in response to inflammation, MSCs form actin- and microtubes-dependent tunneling nanotubes (TNTs) and utilize them for intercellular exchange of content, including organelles, proteins, and mRNA [\\u003cspan additionalcitationids=\\\"CR21\\\" citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. This is possible in the same way for molecular components associated with DUX4 activity. MSCs form TNTs not only among themselves [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e] but also with other cell types, including muscle and muscle-like cells [\\u003cspan additionalcitationids=\\\"CR24\\\" citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], DUX4-expressing cells are able to exert a detrimental effect on neighboring DUX4-negative nuclei and cells in mixed muscle structures, demonstrating the possibility of \\u0026ldquo;spreading\\u0026rdquo; the pathological effect beyond the immediate DUX4-expressing myonuclei [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. To assess the effect of DUX4 expression on MSCs in the context of FSHD, we constructed a cellular model of MSCs with inducible ectopic \\u003cem\\u003eDUX4\\u003c/em\\u003e expression (MSC-DUX4) and analyzed its gene expression and differentiation potential. Surprisingly, we observed that DUX4 expression induced a reduction of adipogenic and osteogenic potential, disruption in extracellular matrix remodeling and angiogenesis factors, but an increased, but limited capacity for myogenic differentiation.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCell cultures\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHuman mesenchymal stromal cells (MSCs) derived from adipose tissue were obtained from the Cell Culture Collection of the IDB RAS, Moscow, Russia. They were cultured in DMEM/F12 (Paneco) supplemented with 10% fetal bovine serum (FBS; HyClone), Glutamax (Gibco), penicillin\\u0026ndash;streptomycin (Gibco), and Insulin-Transferrin-Selenium supplement (ITS; Gibco). Cells are available free of charge upon request from the Cell Culture Collection of the IDB RAS, Moscow, Russia.\\u003c/p\\u003e\\n\\u003cp\\u003eImmortalized myoblasts derived from a healthy individual (AB1190) and from a FSHD patient (AB1080) [28] were a kind gift of Dr. V. Mouly (Institute of Myology, Paris, France). AB1080 and AB1190 were cultured in a proliferation medium composed of four parts of high-glucose DMEM and one part of Medium 199 (Sigma-Aldrich #M4530) supplemented with 20 % FBS (Life technology #10270), 25 \\u0026micro;g/ml Fetuine (Life technology #10344026), 5 ng/ml Human epidermal growth factor (Life technology #PHG0311), 0.5 ng/ml Basic fibroblast growth factor (Life technology #PHG0026), 5 \\u0026micro;g/ml Insulin (Sigma #91077C-1G), 0.2 \\u0026micro;g/ml Dexamethasone (Sigma #D4902), 1 % penicillin\\u0026ndash; streptomycin (Gibco #15140-122). Cells are available free of charge upon request from the Institute of Myology, Paris, France.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eConstruction of the MSC-DUX4 cell line\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe created a model of MSC with inducible DUX4 expression (MSC-DUX4) using the approach described elsewhere [29]. Briefly, the pCW57.1 - DUX4 - WT vector (Addgene plasmid #99282) was transfected into the HEK293T packaging cell line. The viral supernatant was collected 48 and 72 hours post-transfection and filtered. For transduction, MSCs were seeded to form a complete confluent monolayer and then incubated with the lentiviral particles containing the inducible DUX4 construct and a puromycin resistance gene in the presence of polybrene for 24 hours. The following day, the viral-containing medium was replaced with fresh growth medium.\\u003c/p\\u003e\\n\\u003cp\\u003eAfter an additional 24-hour incubation in the growth medium, selection was initiated with 0.75 \\u0026mu;g/mL puromycin and maintained for 5 days, with medium was changed every 48 hours. Puromycin concentration was determined experimentally for MSCs. Following selection, the cells proliferated and formed dense colonies. DUX4 expression was induced by adding 1 \\u0026mu;g/mL doxycycline for 48 hours unless otherwise stated in the text.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eDifferentiation induction\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eMyogenic differentiation\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eMyoblasts\\u0026nbsp;\\u003c/em\\u003ewere seeded at \\u0026gt;95% confluence to form a complete monolayer. After 24 hours, the growth medium was replaced with a differentiation-inducing medium composed of high-glucose DMEM supplemented with 2% horse serum (Paneco), Glutamax, and Penicillin\\u0026ndash;Streptomycin. No medium changes were performed throughout the differentiation period. On day 5, myotubes were stained with May\\u0026ndash;Gr\\u0026uuml;nwald\\u0026ndash;Giemsa.\\u003c/p\\u003e\\n\\u003cp\\u003eMSCs were seeded at \\u0026gt;95% confluence to form a complete monolayer. After 24 hours, the growth medium was replaced with a differentiation-inducing medium composed of high-glucose DMEM supplemented with 2% horse serum, Glutamax, Penicillin-Streptomycin, 1 ng/ml bFGF, hydrocortisone (Sigma) and 0,1 \\u0026micro;M dexamethasone. Medium changes were performed every three days throughout the differentiation period. On day 10, myotube-like structures were stained with May\\u0026ndash;Gr\\u0026uuml;nwald\\u0026ndash;Giemsa.\\u003c/p\\u003e\\n\\u003cp\\u003eMicroscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera. For each sample, at least 10 randomly selected fields were imaged, and measurements were analyzed in ImageJ. Myotube fusion index (FI) were quantified, where FI was calculated as the percentage of nuclei located within myotubes relative to the total number of nuclei.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eOsteogenic differentiation\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMSCs were seeded at \\u0026gt;95% confluence to form a complete monolayer. After 24 hours, the standard growth medium was replaced with an osteogenic induction medium: high-glucose DMEM, 10% FBS, 0,1 \\u0026micro;M dexamethasone, 50 \\u0026micro;M ascorbate-2-phosphate (Sigma), 10 mM \\u0026beta;-glycerophosphate (Sigma). MSCs were cultured for 21 days in the induction medium; the medium was changed every three days during differentiation . Alizarin Red S staining was performed followed by quantitative analysis of mineralization and calcium deposition on day 21 of differentiation. Microscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eAdipogenic differentiation\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMSCs were seeded at \\u0026gt;95% confluence to form a complete monolayer. After 24 hours, the standard growth medium was replaced with an adipogenic medium: high-glucose DMEM, 10% FBS, 3-Isobutyl-1-methylxanthine (IBMX; Sigma), 1 \\u0026micro;M dexamethasone, ITS, 200 \\u0026micro;М indometacin (Sigma). Oil Red O staining was performed followed by quantitative analysis of neutral lipid content on day 21 of differentiation. Microscopic analysis was performed using an Olympus IX51 inverted microscope equipped with an Olympus DP70 camera.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eImmunofluorescence staining\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were rinsed with PBS (Paneco), fixed in 4% paraformaldehyde (PFA) for 5\\u0026ndash;10 minutes at 4 \\u0026deg;C, and incubated for 30 minutes in a blocking buffer containing 0.1% Triton X-100 (Sigma-Aldrich) and 4% FBS in PBS. Primary antibodies \\u003cstrong\\u003e(Table S1)\\u003c/strong\\u003e, diluted in the same blocking buffer, were applied overnight at 4 \\u0026deg;C in a humid chamber, then secondary antibodies \\u003cstrong\\u003e(Table S2)\\u003c/strong\\u003e were added for 1 hour at room temperature (RT). Nuclei were counterstained with DAPI (Sigma). Imaging was performed using an Olympus IX73 inverted fluorescence microscope equipped with an Olympus DP camera. For each sample, at least 10 random fields were captured and analyzed using ImageJ as described elsewhere [18].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMay-Grunwald Giemsa staining\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMSCs or myoblasts were stained with May-Grunwald Giemsa dye as previously described [30] after myogenic differentiation. The cells were washed with PBS, fixed with 100% methanol at + 4\\u0026deg;C for 5-10 minutes and air-dried. Then the cells were incubated in May-Grunwald\\u0026apos;s solution (eosin-methylene blue) (1:3 dilution in 1 mM PBS pH 5,6) (MiniMed) for 20 minutes and Giemsa solution (1:20 dilution in 1mM PBS pH 5,6) (Paneco) for 40 minute and washed with distilled water.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAlizarin Red S staining\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter 21 days of osteogenic differentiation, MSCs were washed with PBS and fixed with 4% PFA for 15 minutes at 4\\u0026deg;C. Cells were then stained with 40 mM Alizarin Red S (Sigma) for 2 minutes at RT, followed by rinsing with 1 mM HCl in 95% ethanol. Imaging was performed using an Olympus IX73 inverted phase microscope equipped with an Olympus DP camera. For quantitative assessment of mineralization, cells were stained with 40 mM Alizarin Red S (ARS) for 20-30 minutes at RT. Subsequently, 10% acetic acid was added, and the samples were incubated on a shaker at RT for 30 minutes. The acid solution containing stained cells was collected using a cell scraper, transferred to tubes, and heated at 85\\u0026deg;C for 10 minutes. After incubation on ice for 5 minutes, the slurry was centrifuged at 20,000 \\u0026times; g for 15 minutes. The supernatant was transferred to a new tube and neutralized with 10% ammonium hydroxide. The neutralized solution was aliquoted in triplicate into a 96-well plate. ARS standards were prepared and aliquoted similarly. Absorbance was measured at 405 nm using a microplate reader (Synergy H1 plate reader, BioTek), and ARS concentration was calculated based on a standard curve generated in Excel.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eOil Red O staining\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter 21 days of adipogenic differentiation, MSCs were washed with PBS and fixed with 4% PFA for 15 minutes at 4\\u0026deg;C. Cells were then briefly treated with 60% isopropanol (Macklin) and stained with 0.5% Oil Red O (Sigma-Aldrich) for 10-15 minutes at RT. Following staining, cells were rinsed with 60% isopropanol and washed gently with distilled water. Nuclei were counterstained with Mayer\\u0026apos;s hematoxylin for 5 minutes. Imaging was performed using an Olympus IX73 inverted microscope equipped with an Olympus DP camera. For quantitative assessment of neutral lipid accumulation, the Oil Red O dye was eluted with 100% isopropanol. The resulting solution was aliquoted in triplicate into 96-well plate, and absorbance was measured at 510 nm using a microplate reader (Synergy H1 plate reader, BioTek).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eFlow cytometry\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were detached with 0.05% trypsin\\u0026ndash;EDTA, washed in PBS by centrifugation at 300 g for 10 minutes, and fixed in Cytofix (BD Biosciences) for 20 minutes at 4 \\u0026deg;C. After another wash with PBS, cells were incubated overnight at 4 \\u0026deg;C with primary antibodies anti-DUX4 (\\u003cstrong\\u003e\\u003cem\\u003eTable S2)\\u003c/em\\u003e\\u003c/strong\\u003e; then appropriate secondary antibodies were applied for 1 hour at RT. Samples were washed in PBS again three times aControl samples without primary antibodies were included to assess nonspecific secondary antibody binding. For flow cytometry analysis of MSCs surface markers, cells were detached using 0.05% trypsin-EDTA and washed in PBS (centrifugation at 1300 rpm for 5 minutes). Subsequently, cells were incubated with conjugated antibodies (anti-CD90, anti-CD73, anti-CD44, anti-CD105; \\u003cstrong\\u003e\\u003cem\\u003eTable S3\\u003c/em\\u003e)\\u003c/strong\\u003e for 1 hour at +4\\u0026deg;C, washed three times, and then fixed with Cytofix for 20 minutes at +4\\u0026deg;C. Following PBS washes three times (centrifugation at 1600 rpm for 5 min at +4\\u0026deg;C) and resuspension in Staining buffer (BD Biosciences). Samples were analyzed on an Attune NxT flow cytometer.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMTT test\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were seeded in 96-well plates (10⁵ cells/well) in the growth medium. After 24 hours MTT (thiazolyl blue tetrazolium bromide; Sigma-Aldrich) was added to the culture medium at a final concentration of 0.5 mg/mL, followed by incubation for 3.5 hours at 37\\u0026deg;C. The medium was then carefully removed, and dimethyl sulfoxide (DMSO; Paneco) was added to solubilize the formazan crystals. The plates were incubated for 15 minutes at RT. The resulting solution was aliquoted in triplicate into a 96-well plate, with pure DMSO used as a blank. Absorbance was measured at 590 nm with a reference filter of 620 nm using a microplate reader (Synergy H1 plate reader, BioTek). Absorbance values are proportional to the number of metabolically active cells. Cell viability was expressed as a percentage relative to the control group, and survival curves were generated based on these measurements in Excel.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eROS Detection Assay\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were seeded in 96-well plates (10⁵ cells/well) in the growth medium and allowed to attach overnight. MSCs were washed with PBS. DCFDA Cellular ROS Detection Assay Kit (Abcam, ab113851) was used according to the manufacturer\\u0026apos;s instructions. Cells were washed with PBS and incubated with 25 \\u0026mu;M DCFDA at 37\\u0026deg;C in the dark. Fluorescence levels were measured in control MSC and Dox-induced MSC-DUX4 at 30, 60, and 120 minutes after DCFDA addition using a microplate reader (excitation wavelength: 485 nm; emission wavelength: 535 nm; green channel) (Varioskan, Thermo Fisher). Fluorescence intensity was proportional to intracellular ROS levels.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCollagen assay\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were seeded in 6-wells plates (5*10\\u003csup\\u003e5\\u003c/sup\\u003e cells) in the growth medium. After 24 hours, the growth medium was replaced with the medium containing 2% FBS and DUX4 expression was induced by adding 1 \\u0026mu;g/mL doxycycline. After five days, collagen content was quantified using the Sircol Soluble Collagen Assay Kit (Biocolor). Cultures were washed with PBS and incubated overnight at 4 \\u0026deg;C in 0.5 M acetic acid with 0.1 mg/ ml pepsin on a rotating platform. Cells were scraped, centrifuged at 3000 g for 10 minutes at 4 \\u0026deg;C, and the medium was collected and cleared by centrifugation at 1500 g for 10 minutes at 4 \\u0026deg;C. The Acid Neutralising Reagent was added to the lysates, followed by the addition of cold Isolation \\u0026amp; Concentration Reagent to each tube. Samples were incubated overnight at 4\\u0026deg;C and then centrifuged at 12,000 \\u0026times; rpm for 10 minutes. The supernatant was removed and Sircol Dye Reagent was added to the pellet. Tubes were capped and mixed by inversion, followed by incubation on a gentle mechanical shaker for 30 minutes to allow formation and precipitation of the collagen-dye complex. After incubation for 30 minutes with gentle shaking, tubes were centrifuged at 12,000 \\u0026times; rpm for 10 minutes. The supernatant was discarded, and the pellet was washed with ice-cold Acid-Salt Wash Reagent to remove unbound dye. Tubes were centrifuged again at 12,000 \\u0026times; rpm for 10 minutes, and the wash reagent was drained. Alkali Reagent was added to dissolve the bound dye, and tubes were vortexed for 5 minutes. The solution was transferred to a 96-well microplate, and absorbance was measured at 555 nm using a microplate reader (Synergy H1 plate reader, BioTek). Absorbance values were measured against a water blank for reagent blanks, standards, and test samples and collagen concentration was calculated based on a standard curve generated in Excel.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRT-qPCR\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTotal RNA was extracted using Quick RNA kit (Zymo research). RNA concentration was measured on a BioPhotometer plus spectrophotometer (Eppendorf). RNA samples were stored at -70\\u0026deg;C. Purified RNA was reverse transcribed using the MMLV RT kit (Eurogen). cDNA samples were stored at -20\\u0026deg;C. cDNA was then mixed with 1 \\u0026mu;l primers (10 mkM final) and 2 \\u0026mu;l 5Х HS-SYBR Green Master mix (ROX) (Eurogen) in a final volume was 10 \\u0026mu;l and analyzed on Light cycler 96 (Roche). Primer sequences are listed in the \\u003cstrong\\u003eTable S3.\\u0026nbsp;\\u003c/strong\\u003eReactions were performed in triplicates. Relative gene expression level (RQ) was calculated using the 2 \\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003e\\u003csup\\u003e\\u0026Delta;\\u0026Delta;\\u003c/sup\\u003e\\u003csup\\u003eCt\\u003c/sup\\u003e quantification method relative to \\u003cem\\u003eGAPDH\\u003c/em\\u003e expression.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRNAseq\\u003c/strong\\u003e\\u003cem\\u003e\\u003cbr\\u003e\\u003c/em\\u003eThe samples were prepared in four biological replicates. The cells were grown as described above and DUX4 expression was induced by adding 1 \\u0026mu;g/mL doxycycline. 48h post-induction, the total RNA was extracted using the Nucleospin RNA isolation kit (Macherey\\u0026ndash;Nagel, 740955) according to the manufacturer\\u0026apos;s protocol. A total amount of 1 \\u0026micro;g RNA per sample was used as input material for the sequencing library preparations. Sequencing libraries were generated using NEBNext\\u0026reg; Ultra TM RNA Library Prep Kit for Illumina\\u0026reg; (NEB, USA) following manufacturer\\u0026rsquo;s recommendations. The library quality was assessed on the Agilent Bioanalyzer 2100 system. Prepared libraries were sequenced on an Illumina HiSeq2000.\\u003c/p\\u003e\\n\\u003cp\\u003eRaw paired-end sequence reads were mapped to the human genome (GRCh38) using STAR (release 2.7.3a) [31]. Reads were assigned to genes via featureCounts (v 2.0.0) [32]. Differential expression analysis was performed using the DESeq2 R package [33]. Genes with |log2Fold change| \\u0026gt; 1 and adjusted p-values( padj) \\u0026lt;0.05 were considered significantly differentially expressed. The results of the differential expression analysis are provided in \\u003cstrong\\u003eTable\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;S5\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eEnrichment analyses were performed against the HALLMARK gene sets of the Molecular Signature Database (https://www.gsea-msigdb.org/gsea/msigdb/) using Gene Set Enrichment Analysis (GSEA). All genes with padj \\u0026lt; 0.05 were used.\\u003c/p\\u003e\\n\\u003cp\\u003eThe gene sets used in the venn diagrams and volcano plots were taken from Molecular Signature Database. The geneset IDs in the MSigDB are mentioned in the corresponding figure legends.\\u003c/p\\u003e\\n\\u003cp\\u003eThe code used to generate the results and figures is deposited to GitHub (https://github.com/annaschwager/MSC_DUX4).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eStatistical analysis\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDepending on data distribution, either Student\\u0026rsquo;s two-tail t-test or the nonparametric Mann\\u0026ndash;Whitney test was applied for pairwise comparisons. For multiple comparisons, the Kruskal\\u0026ndash;Wallis test with FDR correction was used. Analyses were performed in GraphPad Prism 10.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMSC-DUX4 cells inducibly express DUX4\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo model the effect of DUX4 expression on MSCs, we created a cell line with inducible DUX4 expression (MSC-DUX4) as described elsewhere [34] using a lentiviral vector with doxycycline (Dox)-inducible promoter and the puromycin resistance gene (puroR) (see Materials and Methods). DUX4 expression was induced by adding 1 \\u0026mu;g/mL doxycycline and confirmed in MSC-DUX4 cells by immunofluorescence staining (\\u003cstrong\\u003eFigure 1A\\u003c/strong\\u003e), flow cytometry (\\u003cstrong\\u003eFigure 1B\\u003c/strong\\u003e) and PCR analysis \\u003cstrong\\u003e(Figure 1C)\\u003c/strong\\u003e. It should be noted that even without induction, when cultured in commercial serum not certified as tetracycline-free, MSC-DUX4 exhibited low level DUX4 expression due to promoter leakage similarly to MB135-DUX4 cells [35]. This is clearly visible in flow cytometric analysis of \\u003cem\\u003eDUX4 \\u003c/em\\u003eexpression in MSC-DUX4 cells (\\u003cstrong\\u003eFigure 1B\\u003c/strong\\u003e). After confirming the DUX4 construct integration into the MSCs, we performed flow cytometry on Dox-induced MSC-DUX4 to assess the expression of major MSCs markers. Indeed, Dox-induced MSC-DUX4 cells were similar to the control MSCs and did not express HLA-DR. Over 95% of control MSCs were positive for CD73, CD90, and CD105, consistent with international standards for MSCs identification [36]. Likewise, over 95% of Dox-induced MSC-DUX4 were positive for CD90, ~ 93% for CD73, and ~ 90% for CD105. These results indicate that Dox-induced MSC-DUX4 retained positive expression of the core MSCs markers (CD73, CD90, CD105) as well as the additional marker CD44, while maintaining negative expression of HLA-DR, a phenotype identical to that of the control MSCs (\\u003cstrong\\u003eFigure 1C\\u003c/strong\\u003e). Similar to control MSCs, Dox-induced MSC-DUX4 cells were consistently negative for CD56, confirming the absence of a myogenic phenotype (\\u003cstrong\\u003eFigure S1AB\\u003c/strong\\u003e).\\u003c/p\\u003e\\n\\n\\u003cp\\u003eDUX4 induction in MB135-DUX myoblasts triggers massive apoptosis within 48 hours [34, 35]. We assessed the viability of MSC-DUX4 cells after DUX4 induction using the MTT assay for 8 days after DUX4 induction \\u003cstrong\\u003e(Figure 1D)\\u003c/strong\\u003e. In contrast to MB135-DUX4 myoblasts which underwent cell death within 48 hours, MSC-DUX4 cells retained viability for at least eight days after DUX4 induction. \\u003c/p\\u003e\\n\\n\\u003cp\\u003eAs elevated DUX4 expression induces oxidative stress in myoblasts [34, 37, 38], we analyzed intracellular reactive oxygen species (ROS) in Dox-induced MSC-DUX4 cells using DCFDA which diffuses into cells and is oxidized to fluorescent DCF in the presence of ROS. We found that after induction of the DUX4 expression, MSCs accumulated significantly more ROS \\u003cstrong\\u003e(Figure 1E)\\u003c/strong\\u003e. Real-time impedance-based monitoring with the xCelligence system revealed an approximately two-fold reduction in proliferation rate in MSCs upon induction of DUX4 expression (\\u003cstrong\\u003eFigure S1C\\u003c/strong\\u003e). \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAnalysis of gene expression pattern induced in MSC by DUX4\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSimilarly to myoblasts, MSCs of FSHD patients can also express DUX4 [39], although the effect of DUX4 on MSCs gene expression has never been evaluated. To screen for the MSC-expressed factors that may contribute to muscle impairment in FSHD, we performed RNA-seq in the Dox-induced MSC-DUX4 and the control MSCs (\\u003cstrong\\u003eFigure 2\\u003c/strong\\u003e). We observed the upregulation (log2Fold \\u0026gt;1, padj \\u0026lt; 0.05) of 1716 and downregulation (log2Fold \\u0026lt; -1, padj \\u0026lt; 0.05) of 1356 genes in MSC-DUX4 cells induced for \\u003cem\\u003eDUX4\\u003c/em\\u003e expression compared to the control MSC cells \\u003cstrong\\u003e(Table S5)\\u003c/strong\\u003e. As expected, we detected the upregulation of several known DUX4 targets, e.g. \\u003cem\\u003eZSCAN4\\u003c/em\\u003e and \\u003cem\\u003eTRIM43 \\u003c/em\\u003e(\\u003cstrong\\u003eFigure 2A, B; Figure S2A\\u003c/strong\\u003e), as well as the downregulation of the PAX7 signature, another common trait of \\u003cem\\u003eDUX4\\u003c/em\\u003e-expressing cells [40] (\\u003cstrong\\u003eFigure 2C, D; Figure S2B\\u003c/strong\\u003e).\\u003c/p\\u003e\\n\\n\\u003cp\\u003eTo screen for the biological themes consistently deregulated in MSC by DUX4 expression, we have performed Gene Set Enrichment Analysis (GSEA) against the hallmark gene sets of the Molecular Signatures Database (MSigDB) (\\u003cstrong\\u003eFigure 2E\\u003c/strong\\u003e). The strongest upregulated pathways turned out to be related to the inflammatory response. Among upregulated genes, \\u003cem\\u003eIL6, TNF, CCL2/5/7/20, CXCL1/6/10/11, IL1A, IL15,\\u003c/em\\u003e belong to a chemotactic and pro-inflammatory secretome indicating an increased capacity of DUX-expressing MSCs to recruit and activate monocytes, neutrophils, T cells and NK cells as well as promote local inflammation. We have also detected a strong downregulation of the genes related to cell division and cell cycle (\\u003cstrong\\u003eFigure 2E\\u003c/strong\\u003e), consistent with a prominent decrease in the doubling time of the Dox-induced MSC-DUX4 cells (\\u003cstrong\\u003eFigure S1C\\u003c/strong\\u003e).\\u003cbr\\u003e \\u003c/p\\u003e\\n\\u003cp\\u003eTo assess the effect of DUX4 expression on the lineage commitment of the MSCs, we have evaluated the expression of the genes involved in the major MSC differentiation pathways, osteogenic, adipogenic, chondrogenic and myogenic. DUX4 expression resulted in the deregulation of the genes involved in all of these differentiation strategies (\\u003cstrong\\u003eFigure 2F\\u003c/strong\\u003e). Notably, we were intrigued by a relatively large number (30) of upregulated genes in the HALLMARK:MYOGENESIS gene set (\\u003cstrong\\u003eFigure 2F\\u003c/strong\\u003e). Those genes encoded for some of the key actors or markers of muscle development, e.g. \\u003cem\\u003eTNNT1\\u003c/em\\u003e (4.3 log2fold, 1.5e-70 padj) - a troponin subunit essential for striated muscle contraction, \\u003cem\\u003eDMD\\u003c/em\\u003e (1.3 log2fold, 3.9e-37 padj) - a crucial muscle protein connecting the cytoskeleton of a muscle fiber to the extracellular matrix, and \\u003cem\\u003eSVIL \\u003c/em\\u003e(1.6 log2fold, 3.4e-98 padj) - one of the first costameric proteins to assemble during myogenesis (\\u003cstrong\\u003eFigure 2G\\u003c/strong\\u003e). We have also observed the downregulation of some genes related to the negative regulation of smooth muscle cell differentiation (\\u003cstrong\\u003eFigure S2\\u003c/strong\\u003e). These results suggested that DUX4-expressing MSC might acquire myogenic capacities. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eDUX4 confers a limited myogenic potential to mesenchymal stem cells\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo experimentally test our prediction that DUX4 expression conferred a myogenic potential to MSC-DUX4 cells, we induced myogenic differentiation in the control MSCs and Dox-induced MSC-DUX4 as described elsewhere [18]. In contrast to the control MSCs, Dox-induced MSC-DUX4 cells formed myotube-like structures on day 10 of differentiation whereas normal human myoblasts show this pattern at days 4-6 [18, 35] \\u003cstrong\\u003e(Figure S3A)\\u003c/strong\\u003e. Myotube-like structures formed by MSC-DUX4 cells were positive for desmin, MF20 (anti-MYH), Troponin T and myogenin. MF20, myogenin, Troponin T are markers of mature myotubes, desmin is a marker for both committed and differentiating myoblasts. We observed that, in contrast to control MSC, Dox-induced MSC-DUX4 cells formed myotube-like structures and expressed key myogenic markers. Immunostaining was positive for MF20, troponin T (markers of late myogenesis;\\u003cstrong\\u003e Figure 3A\\u003c/strong\\u003e), and myogenin (a marker of mid myogenesis; \\u003cstrong\\u003eFigure S3B\\u003c/strong\\u003e). Furthermore, Dox-induced MSC-DUX4 cells expressed desmin, another early marker of myogenic commitment (\\u003cstrong\\u003eFigure S3B\\u003c/strong\\u003e). Control MSC also exhibited a positive staining for desmin, consistent with its role as an early differentiation marker and its known expression in committed myogenic progenitors. It should be noted that neither control MSCs nor MSC-DUX4 were positive for CD56, a marker of myogenic progenitor cells (\\u003cstrong\\u003eFigure S1AB\\u003c/strong\\u003e). To quantify the efficiency of myotube formation, we calculated the fusion index (FI) defined as the number of nuclei inside the myotubes divided by the total number of nuclei * 100%. The FI of Dox-induced MSC-DUX4 cells was approximately 10-fold lower than that of the control myoblasts, even though the Dox-induced MSC-DUX4 cells were capable of generating myotube-like structures contrary to the control MSCs \\u003cstrong\\u003e(Figure 3B)\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\n\\u003cp\\u003eWe next directly compared the expression of myogenic markers (myogenin, desmin and troponin T) in Dox-induced MSC-DUX4 to myoblasts on day 5 of differentiation using quantitative real-time PCR (RT-qPCR).The expression levels of myogenesis markers in Dox-induced MSC-DUX4 were significantly lower or entirely absent compared to even FSHD myotubes \\u003cstrong\\u003e(Figure 3C)\\u003c/strong\\u003e, suggesting that Dox-induced MSC-DUX4 derived myotubes might be functionally defective. Expression levels of Troponin T2 were 0.00002 \\u0026plusmn; 0.0 vs. 0.00006 \\u0026plusmn; 0. 00001 vs. 1.30134 \\u0026plusmn; 0.06505 for MSCs, Dox-induced MSC-DUX4 and FSHDmyoblasts respectively; expression levels of myogenin were: 0 vs. 0 vs. 0.13584 \\u0026plusmn; 0.00942 for MSCs and Dox-induced MSC-DUX4 and FSHD-myoblasts respectively; expression levels of desmin: 0 vs 0.00035 \\u0026plusmn; 0.00003 vs 1.85318 \\u0026plusmn; 0.12845 for MSCs and Dox-induced MSC-DUX4 and FSHD-myoblasts, respectively. We thus made an exciting discovery that Dox-induced MSC-DUX4 were to some extent capable of forming myotubes, in contrast to the control MSC. However, these structures may not represent mature, functional myotubes comparable to those derived from \\u003cem\\u003ebona fide\\u003c/em\\u003e myoblasts.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eDUX4 reduces the capacity of MSC for the adipogenic and osteogenic differentiation\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNext, we compared differentiation capacities of the control MSC and Dox-induced MSC-DUX4 for the adipogenic and osteogenic differentiation. MSCs were cultured for 21 days in either osteogenic or adipogenic induction medium as described in the Material and Methods. Osteogenic differentiation was evaluated by Alizarin Red S staining followed by quantitative analysis of extracellular matrix mineralization (calcium deposition) as described in Material and Methods \\u003cstrong\\u003e(Figure 4A)\\u003c/strong\\u003e. Adipogenic differentiation was evaluated by Oil Red O staining followed by quantitative analysis of neutral lipid content as described in Material and Methods \\u003cstrong\\u003e(Figure 4B)\\u003c/strong\\u003e. In contrast to the control MSC, Dox-induced MSC-DUX4 cells had a reduced capacity for osteogenic and adipogenic differentiation. Dox-induced MSC-DUX4 accumulated approximately four-fold less lipids (0.16 \\u0026plusmn; 0.01 vs. 0.68 \\u0026plusmn; 0.03 OD\\u003csub\\u003e510\\u003c/sub\\u003e) compared to the control MSC (\\u003cstrong\\u003eFigure 4A\\u003c/strong\\u003e) and approximately two times less calcium 3.54 \\u0026plusmn; 0.56 vs.(7.3 \\u0026plusmn; 1.7 mM) (\\u003cstrong\\u003eFigure 4B\\u003c/strong\\u003e). \\u003c/p\\u003e\\n\\u003cp\\u003eThe expression of an early adipogenic marker FABP4 and a late marker leptin was analyzed by immunostaining (\\u003cstrong\\u003eFigure S4A\\u003c/strong\\u003e) and RT-qPCR (\\u003cstrong\\u003eFigure S4B\\u003c/strong\\u003e) on the 5th, 15th and 21th day of differentiation. FABP4 was expressed at high levels on day 5 of differentiation in the control MSCs. Its expression then decreased approximately two-fold and remained stable until day 21. Leptin expression in the control MSCs exhibited a wave-like pattern: it increased by day 5, declined to undetectable levels by day 15, and increased again by day 21. In contrast, Dox-induced MSC-DUX4 showed markedly altered expression profiles. First, the overall expression level of FABP4 was two times lower compared to the controls. The dynamics of FABP4 expression was also different: instead of peaking on day 5, expression continued to increase until day 15, indicating a clear temporal shift in the differentiation. Leptin expression was nearly absent in Dox-induced MSC-DUX4, suggesting a failure to undergo terminal adipogenic differentiation. Together, these results indicate that high levels of DUX4 expression impair the adipogenic differentiation potential of MSCs.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eDUX4 induces pro-fibrotic activity of MSCs\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe have previously demonstrated that MSCs could produce and secrete extracellular matrix proteins, such as collagen and fibronectin, thereby contributing to fibrosis development in the context of FSHD [18]. Here we assessed the contribution of DUX4-expressing MSC to the extracellular matrix (ECM) production. \\u003c/p\\u003e\\n\\n\\u003cp\\u003eWe have observed transcriptional upregulation of 45 genes comprising the matrisome in the Dox-induced MSC-DUX4 cells, including 8 collagen-encoding genes and 35 genes encoding structural ECM glycoproteins (\\u003cstrong\\u003eFigure 5AB\\u003c/strong\\u003e). We have thus experimentally evaluated the collagen synthesis and secretion of collagen, a major ECM protein in the DUX4-expressing and control MSC. Control MSC and Dox-induced MSC-DUX4 were immunocytochemically stained for collagen types III and IV (\\u003cstrong\\u003eFigure 5C\\u003c/strong\\u003e). Secreted collagen appears as thin filaments in the extracellular space(yellow arrows in \\u003cstrong\\u003eFigure 5C\\u003c/strong\\u003e). We found that Dox-induced MSC-DUX4 exhibited an increased level of collagen types III and IV secretion compared to the control MSC. Next, we quantified collagen accumulation in lysates of the control and Dox-induced MSC-DUX4 using Sircol Soluble Collagen Assay Kit . Collagen concentration in lysates was measured five days post-DUX4 induction. We detected a two-fold increase in collagen content in Dox-induced MSC-DUX4 lysates compared to the control MSC \\u003cstrong\\u003e(Figure 5D)\\u003c/strong\\u003e. Additionally, we stained the control and Dox-induced MSC-DUX4 with antibodies against an ECM glycoprotein fibronectin and observed that Dox-induced MSC-DUX4 secreted thicker fibronectin fibers compared to the control \\u003cstrong\\u003e(Figure 5E).\\u003c/strong\\u003e\\u003c/p\\u003e\\n\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eFacioscapulohumeral muscular dystrophy (FSHD) is characterized both by skeletal muscle degeneration and progressive replacement of muscle tissue with fibrotic and adipose tissue [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. The skeletal muscle niche harbors a heterogeneous population of mesenchymal stem cells (MSCs), including both resident MSCs and those migrating to the injured site from other adjacent tissues following muscle injury [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]; they become part of the fibroadipogenic precursor (FAP) compartment. Consequently, the FAP population in regenerating muscle includes both resident FAP and infiltrated cells [\\u003cspan additionalcitationids=\\\"CR15\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. In this study, we specifically concentrate on cells migrating into muscle, particularly from adipose tissue. They rapidly infiltrate damaged muscle and significantly contribute to tissue regeneration and remodeling. Recent studies indicate that these mobilized MSCs affect myogenesis [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eSince contraction of the 4q35 D4Z4 array or its derepression leading to DUX4 expression is a hallmark of FSHD [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], reviewed in [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], we hypothesized that MSCs recruited to sites of injury and involved in tissue repair are also exposed to the pathogenic DUX4 signal. This hypothesis is based on evidence that, in response to inflammation, MSCs form actin- and microtubule-dependent tunneling nanotubes (TNTs) and utilize them for intercellular exchange of content, including organelles, proteins, and mRNA [\\u003cspan additionalcitationids=\\\"CR22\\\" citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. This is possible in the same way for molecular components associated with DUX4 activity. MSCs form TNTs not only among themselves [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] but also with other cell types, including muscle and muscle-like cells [\\u003cspan additionalcitationids=\\\"CR25\\\" citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e], making TNT-mediated communication with myoblasts highly plausible and creating a route for the intercellular transfer of molecular components associated with DUX4 activity. In addition, DUX4-expressing cells are able to exert a detrimental effect on neighboring DUX4-negative nuclei and cells in mixed muscle structures, demonstrating the possibility of \\u0026ldquo;spreading\\u0026rdquo; the pathological effect beyond the immediate DUX4-expressing myonuclei [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Together, these observations support the development of the MSC-DUX4 cellular model to investigate the contribution of DUX4 expression in non-myoblasts cell types involved in muscle regeneration and their intercellular communication in the pathogenesis of FSHD.\\u003c/p\\u003e \\u003cp\\u003eFollowing doxycycline induction, MSC-DUX4 cells robustly expressed DUX4 protein while retaining their characteristic MSC phenotype, including positive expression of CD73, CD90, and CD105, and the absence of HLA-DR and CD56. This stability in surface marker expression confirms that DUX4 induction does not compromise mesenchymal identity. However, functional assays revealed a pronounced reduction in proliferation rate and a marked accumulation of reactive oxygen species (ROS), indicating that DUX4 imposes cellular stress in MSCs similar to that seen in myoblasts [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e] This validates our system as a suitable model for dissecting DUX4-related mechanisms in a mesenchymal context.\\u003c/p\\u003e \\u003cp\\u003eAn important difference emerged when comparing the survival of MSC-DUX4 to previously reported myoblast models ectopically expressing DUX4. While myoblasts rapidly undergo apoptosis upon DUX4 activation [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e], MSC-DUX4 cells remain viable for at least eight days post-induction, suggesting intrinsic differences in stress tolerance and survival mechanisms between these cell types. This differential sensitivity may reflect the greater adaptability of MSCs to oxidative and metabolic stress, consistent with their role as stromal support cells in variable tissue environments [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTranscriptomic analysis revealed extensive gene deregulation upon DUX4 induction, with more than 1,700 upregulated and 1,300 downregulated transcripts. The upregulation of canonical DUX4 targets, such as \\u003cem\\u003eZSCAN4\\u003c/em\\u003e and \\u003cem\\u003eTRIM43\\u003c/em\\u003e, validates the functionality of the system. Conversely, downregulation of \\u003cem\\u003ePAX7\\u003c/em\\u003e target genes previously identified as a hallmark of DUX4 activity [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e] was also evident, confirming that the DUX4 transcriptional network was faithfully recapitulated in MSCs. Gene set enrichment analysis (GSEA) revealed strong induction of inflammatory and immune response pathways, including the upregulation of \\u003cem\\u003eIL6\\u003c/em\\u003e, \\u003cem\\u003eTNF\\u003c/em\\u003e, \\u003cem\\u003eCCL2/5/7/20\\u003c/em\\u003e, and \\u003cem\\u003eCXCL1/6/10/11\\u003c/em\\u003e, all components of a potent pro-inflammatory secretome. These changes imply that DUX4-expressing MSCs could serve as active mediators of local inflammation and immune cell recruitment in FSHD muscle, an emerging theme in recent studies of disease progression.\\u003c/p\\u003e \\u003cp\\u003eAnother finding was the significant transcriptional suppression of genes related to cell cycle and proliferation, in agreement with the observed reduction in doubling time. This suggests that while DUX4-expressing MSCs remain viable, their proliferative capacity is impaired, potentially limiting their regenerative contributions within muscle tissue. Interestingly, DUX4 also disrupted lineage-specific gene programs, simultaneously affecting osteogenic, adipogenic, chondrogenic, and myogenic differentiation pathways. Particularly noteworthy was the enrichment of the HALLMARK:MYOGENESIS gene set, including upregulation of \\u003cem\\u003eTNNT1\\u003c/em\\u003e, \\u003cem\\u003eDMD\\u003c/em\\u003e, and \\u003cem\\u003eSVIL\\u003c/em\\u003e, as well as the downregulation of negative regulators of myogenesis. These transcriptional changes predicted an unexpected partial acquisition of myogenic traits by MSC-DUX4.\\u003c/p\\u003e \\u003cp\\u003eIndeed, functional differentiation assays confirmed that DUX4 expression conferred limited myogenic capacity to MSCs. Dox-induced MSC-DUX4 cells formed myotube-like structures expressing desmin, myogenin, troponin T, and MYH, markers characteristic of various stages of myogenesis. However, these myotube-like structures were sparse and morphologically immature, and their fusion index was approximately ten times lower than that of normal myoblasts. Quantitative PCR further revealed low or absent expression of myogenic markers compared even with FSHD myotubes, suggesting that while DUX4 can initiate partial myogenic reprogramming, it cannot sustain a complete or functional myogenic differentiation program in MSCs. This partial reprogramming may reflect a transient activation of myogenic transcriptional modules by DUX4, insufficient for terminal differentiation.\\u003c/p\\u003e \\u003cp\\u003eIn parallel, DUX4 impaired the canonical differentiation potential of MSCs. Both osteogenesis and adipogenesis were markedly reduced, with disrupted temporal dynamics of adipogenic markers (FABP4, leptin). These findings suggest that DUX4 reprograms MSCs fate, diverting them away from their physiological lineages and potentially altering tissue homeostasis. Conversely, reductions in whole-body and regional bone mineral density (BMD) are associated with diminished muscle strength and functional capacity in FSHD patients who also show an increased frequency of traumatic fractures [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eOne of the most striking effects of DUX4 induction was the enhancement of pro-fibrotic activity. DUX4-expressing MSCs showed transcriptional upregulation of numerous matrisome genes, including those encoding collagens and structural ECM components. Immunocytochemical and biochemical assays confirmed elevated synthesis and secretion of collagen types III and IV, as well as thickened fibronectin fibers. Given the established role of MSCs in fibrosis in the FSHD context [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], this effect provides a compelling mechanism by which DUX4 expression in MSCs could exacerbate the fibrotic environment characteristic of FSHD muscle [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e].This is consistent with the described upregulation of fibroadipogenic progenitor gene expression in samples from FSHD patients and model organisms [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTaken together, our findings redefine the role of DUX4 in FSHD pathology by demonstrating that MSCs, in addition to myoblasts, may represent an additional target of DUX4-mediated reprogramming. In MSCs, DUX4 promotes oxidative stress, inflammation, and fibrosis while impairing normal differentiation and regeneration. The partial acquisition of myogenic traits coupled with a pro-fibrotic secretory profile suggests that MSC-DUX4 may contribute to the aberrant tissue remodeling characteristic of FSHD. However in our system, the apparent concurrent induction of myogenic and profibrotic programs likely reflects cell‑to‑cell heterogeneity of DUX4 levels rather than their co‑activation in the same cells. These processes are spatially segregated across different subpopulations. Endogenous DUX4 expression in FSHD myogenic cells is well known to be rare, stochastic and heterogeneous, with only a small fraction of nuclei/cells showing a high DUX4/DUX4‑target signature at any given time [\\u003cspan additionalcitationids=\\\"CR51\\\" citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. RNA-seq and MERFISH show the separation of \\u0026ldquo;FSHD-hghi\\u0026rdquo; and \\u0026ldquo;FSHD-low\\u0026rdquo; clusters/states by DUX4 targets, i.e., transcriptional heterogeneity within a single culture and even a single myotube, and the FSHD profile is determined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses [\\u003cspan additionalcitationids=\\\"CR54\\\" citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. A similar pattern is observed in our MSC‑DUX4 model (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). At high DUX4 levels, apoptosis and severe differentiation impairments predominate [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. At lower DUX4 levels, myogenesis is impaired without an immediate cell death [\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e]. Even \\\"transient DUX4 bursts\\\" leave a persistent profibrotic state of FAP-like cells and an increased susceptibility to fibrosis [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. Therefore, we propose that MSCs with low or absent expression follow the canonical adipogenic trajectory for MSCs, while DUX4-positive cells form defective myotubes and/or activate profibrotic transcriptional programs.\\u003c/p\\u003e \\u003cp\\u003eFuture work should aim to determine whether these DUX4-induced alterations in MSC behavior influence muscle repair and intercellular communication in vivo, and whether targeting DUX4 signaling in stromal cells could mitigate fibrotic progression and improve muscle function in FSHD.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eARS: Alizarin Red S\\u003c/p\\u003e\\n\\u003cp\\u003eBCA: Bicinchoninic Acid Assay\\u003c/p\\u003e\\n\\u003cp\\u003eBMD: Bone Mineral Density\\u003c/p\\u003e\\n\\u003cp\\u003eC2C12: Mouse Myoblast Cell Line\\u003c/p\\u003e\\n\\u003cp\\u003eCCL2: C-C Motif Chemokine Ligand 2\\u003c/p\\u003e\\n\\u003cp\\u003eCD: Cluster of Differentiation\\u003c/p\\u003e\\n\\u003cp\\u003eCXCL1: C-X-C Motif Chemokine Ligand 1\\u003c/p\\u003e\\n\\u003cp\\u003eCXCL12: C-X-C Motif Chemokine Ligand 12\\u003c/p\\u003e\\n\\u003cp\\u003eCXCR4: C-X-C Chemokine Receptor 4\\u003c/p\\u003e\\n\\u003cp\\u003eD4Z4: D4Z4 Macrosatellite Repeat on Chromosome 4\\u003c/p\\u003e\\n\\u003cp\\u003eDAPI: 4\\u0026prime;,6-Diamidino-2-Phenylindole\\u003c/p\\u003e\\n\\u003cp\\u003eDCF: Dichlorofluorescein\\u003c/p\\u003e\\n\\u003cp\\u003eDCFDA: 2\\u0026prime;,7\\u0026prime;-Dichlorodihydrofluorescein Diacetate\\u003c/p\\u003e\\n\\u003cp\\u003eDMEM: Dulbecco\\u0026rsquo;s Modified Eagle Medium\\u003c/p\\u003e\\n\\u003cp\\u003eDMSO: Dimethyl Sulfoxide\\u003c/p\\u003e\\n\\u003cp\\u003eDNA: Deoxyribonucleic Acid\\u003c/p\\u003e\\n\\u003cp\\u003eDPBS: Dulbecco\\u0026rsquo;s Phosphate-Buffered Saline\\u003c/p\\u003e\\n\\u003cp\\u003eDUX4: Double Homeobox 4\\u003c/p\\u003e\\n\\u003cp\\u003eECL: Enhanced Chemiluminescence\\u003c/p\\u003e\\n\\u003cp\\u003eECM: Extracellular Matrix\\u003c/p\\u003e\\n\\u003cp\\u003eEDTA: Ethylenediaminetetraacetic Acid\\u003c/p\\u003e\\n\\u003cp\\u003eFABP4: Fatty Acid Binding Protein 4\\u003c/p\\u003e\\n\\u003cp\\u003eFAP: Fibro-Adipogenic Progenitor\\u003c/p\\u003e\\n\\u003cp\\u003eFBS: Fetal Bovine Serum\\u003c/p\\u003e\\n\\u003cp\\u003eFDR: False Discovery Rate\\u003c/p\\u003e\\n\\u003cp\\u003eFSHD: Facioscapulohumeral Muscular Dystrophy\\u003c/p\\u003e\\n\\u003cp\\u003eFSHD1: Facioscapulohumeral Muscular Dystrophy Type 1\\u003c/p\\u003e\\n\\u003cp\\u003eFSHD2: Facioscapulohumeral Muscular Dystrophy Type 2\\u003c/p\\u003e\\n\\u003cp\\u003eGAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase\\u003c/p\\u003e\\n\\u003cp\\u003eGSEA: Gene Set Enrichment Analysis\\u003c/p\\u003e\\n\\u003cp\\u003eH3K9: Histone H3 Lysine 9\\u003c/p\\u003e\\n\\u003cp\\u003eHEK293T: Human Embryonic Kidney 293T Cells\\u003c/p\\u003e\\n\\u003cp\\u003eHLA-DR: Human Leukocyte Antigen DR\\u003c/p\\u003e\\n\\u003cp\\u003eIBMX: 3-Isobutyl-1-Methylxanthine\\u003c/p\\u003e\\n\\u003cp\\u003eIL: Interleukin\\u003c/p\\u003e\\n\\u003cp\\u003eITS: Insulin-Transferrin-Selenium Supplement\\u003c/p\\u003e\\n\\u003cp\\u003eMERFISH: Multiplexed Error-Robust Fluorescence In Situ Hybridization\\u003c/p\\u003e\\n\\u003cp\\u003eMMLV: Moloney Murine Leukemia Virus\\u003c/p\\u003e\\n\\u003cp\\u003eMRI: Magnetic Resonance Imaging\\u003c/p\\u003e\\n\\u003cp\\u003eMSC: Mesenchymal Stromal Cell\\u003c/p\\u003e\\n\\u003cp\\u003eMTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide\\u003c/p\\u003e\\n\\u003cp\\u003eMYH: Myosin Heavy Chain\\u003c/p\\u003e\\n\\u003cp\\u003eNMD: Nonsense-Mediated mRNA Decay\\u003c/p\\u003e\\n\\u003cp\\u003ePBS: Phosphate-Buffered Saline\\u003c/p\\u003e\\n\\u003cp\\u003ePBST: Phosphate-Buffered Saline with Tween-20\\u003c/p\\u003e\\n\\u003cp\\u003ePCR: Polymerase Chain Reaction\\u003c/p\\u003e\\n\\u003cp\\u003ePFA: Paraformaldehyde\\u003c/p\\u003e\\n\\u003cp\\u003ePAX7: Paired Box Protein 7\\u003c/p\\u003e\\n\\u003cp\\u003eROS: Reactive Oxygen Species\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eEthics approval and consent to participate\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAvailability of data and materials: Data and material will be made available on reasonable request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eThe authors declare that they have no competing interests\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by FSHD Society (USA) and the IDB RAS Government basic research program (0088-2024-0010) to YV and \\u0026nbsp;the Russian Science Foundation Grant No. 25-15-00443 to ED.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYV and EK devised the experiments; OS, AS, EK and ED carried out the experiments; \\u0026nbsp;OS, AS, EK, EV and YV analyzed data, OS, AS, EK and YV \\u0026nbsp;wrote the main manuscript text and prepared figures; YV and ED obtained funding. All authors reviewed and approved the manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll relevant data are within the paper and the Supplementary Data. The code used to generate the results and figures is deposited to GitHub (https://github.com/annaschwager/MSC_DUX4)..\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eMostacciuolo M, Pastorello E, Vazza G, Miorin M, Angelini C, Tomelleri G, Galluzzi G, Trevisan C (2009) Facioscapulohumeral muscular dystrophy: epidemiological and molecular study in a north‐east Italian population sample. 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Dev Cell 22:38\\u0026ndash;51. https://doi.org/10.1016/j.devcel.2011.11.013 \\u003c/li\\u003e\\n\\u003cli\\u003eBosnakovski D, Chan SSK, Recht OO, Hartweck LM, Gustafson CJ, Athman LL, Lowe DA, Kyba M (2017) Muscle pathology from stochastic low level DUX4 expression in an FSHD mouse model. Nat Commun 8:550. https://doi.org/10.1038/s41467-017-00730-1 \\u003c/li\\u003e\\n\\u003cli\\u003eMaiullari S, Mele G, Calandra P, Di Blasio G, Valentini S, Torcinaro A, Manni I, Teveroni E, Mancino F, Proietti L, Maiullari F, Pesavento M, Giorgini L, Putti S, Rizzi R, Bortolani S, Scavizzi F, Raspa M, Ricci E, Piaggio G, Gargioli C, Pontecorvi A, Luvisetto S, Mazzone M, Deidda G, Moretti F (2025) Estrogen rescues muscle regeneration impaired by DUX4 in a humanized xenograft mouse model. 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Front Immunol 12:626755. https://doi.org/10.3389/fimmu.2021.626755 \\u003c/li\\u003e\\n\\u003cli\\u003eSagaradze GD, Basalova NA, Efimenko AYu, Tkachuk VA (2020) Mesenchymal Stromal Cells as Critical Contributors to Tissue Regeneration. Front Cell Dev Biol 8:576176. https://doi.org/10.3389/fcell.2020.576176 \\u003c/li\\u003e\\n\\u003cli\\u003eChagarlamudi H, Corbett A, Stoll M, Bibat G, Grosmann C, Matichak Stock C, Stinson N, Shapiro J, Wagner KR (2017) Bone health in facioscapulohumeral muscular dystrophy: A cross-sectional study. Muscle Nerve 56:1108\\u0026ndash;1113. https://doi.org/10.1002/mus.25619 \\u003c/li\\u003e\\n\\u003cli\\u003eRagozzino E, Bortolani S, Di Pietro L, Papait A, Parolini O, Monforte M, Tasca G, Ricci E (2023) Muscle fibrosis as a prognostic biomarker in facioscapulohumeral muscular dystrophy: a retrospective cohort study. Acta Neuropathol Commun 11:165. https://doi.org/10.1186/s40478-023-01660-4 \\u003c/li\\u003e\\n\\u003cli\\u003eBosnakovski D, Oyler D, Mitanoska A, Douglas M, Ener ET, Shams AS, Kyba M (2022) Persistent Fibroadipogenic Progenitor Expansion Following Transient DUX4 Expression Provokes a Profibrotic State in a Mouse Model for FSHD. Int J Mol Sci 23:1983. https://doi.org/10.3390/ijms23041983 \\u003c/li\\u003e\\n\\u003cli\\u003eHaynes P, Bomsztyk K, Miller DG (2018) Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele. Epigenetics Chromatin 11:47. https://doi.org/10.1186/s13072-018-0215-z \\u003c/li\\u003e\\n\\u003cli\\u003eRickard AM, Petek LM, Miller DG (2015) Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways. Hum Mol Genet 24:5901\\u0026ndash;5914. https://doi.org/10.1093/hmg/ddv315 \\u003c/li\\u003e\\n\\u003cli\\u003eJiang S, Williams K, Kong X, Zeng W, Nguyen NV, Ma X, Tawil R, Yokomori K, Mortazavi A (2020) Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. PLOS Genet 16:e1008754. https://doi.org/10.1371/journal.pgen.1008754 \\u003c/li\\u003e\\n\\u003cli\\u003eZheng D, Wondergem A, Kloet S, Willemsen I, Balog J, Tapscott SJ, Mahfouz A, Van Den Heuvel A, Van Der Maarel SM (2024) snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis. Hum Mol Genet 33:284\\u0026ndash;298. https://doi.org/10.1093/hmg/ddad186 \\u003c/li\\u003e\\n\\u003cli\\u003eWilliams K, Kong X, Zeng W, Nguyen NV, Ma X, Tawil R, Yokomori K, Mortazavi A (2020) Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. 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Int J Mol Sci 23:1983. https://doi.org/10.3390/ijms23041983 \\u003c/li\\u003e\\n\\u003cli\\u003eKnopp P, Krom YD, Banerji CRS, Panamarova M, Moyle LA, den Hamer B, van der Maarel SM, Zammit PS (2016) DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis. J Cell Sci 129:3816\\u0026ndash;3831. https://doi.org/10.1242/jcs.180372 \\u003c/li\\u003e\\n\\u003cli\\u003eBosnakovski D, Xu Z, Ji Gang E, Galindo CL, Liu M, Simsek T, Garner HR, Agha‐Mohammadi S, Tassin A, Copp\\u0026eacute;e F, Belayew A, Perlingeiro RR, Kyba M (2008) An isogenetic myoblast expression screen identifies DUX4‐mediated FSHD‐associated molecular pathologies. EMBO J 27:2766\\u0026ndash;2779. https://doi.org/10.1038/emboj.2008.201 \\u003c/li\\u003e\\n\\u003cli\\u003eBosnakovski D, Gearhart MD, Toso EA, Ener ET, Choi SH, Kyba M (2018) Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression. Sci Rep 8:16957. https://doi.org/10.1038/s41598-018-35150-8 \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"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\":\"info@researchsquare.com\",\"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\":\"DUX4, FSHD, Mesenchymal stem cells, differentiation, FAP\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9094205/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9094205/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eWe developed a cellular model of mesenchymal stem cells (MSCs) with inducible DUX4 expression (MSC-DUX4) to investigate the potential role of MSCs in facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression was successfully induced, with MSC-DUX4 maintaining the characteristic surface marker profile of MSCs. Unlike myoblasts, which rapidly undergo apoptosis upon DUX4 induction, MSC-DUX4 remained viable although they exhibited increased reactive oxygen species (ROS) accumulation. Transcriptomic analysis revealed broad changes, including strong upregulation of several myogenic genes, suggesting that DUX4 confers a partial myogenic program to MSCs. Indeed, Dox-induced MSC-DUX4 formed myotube-like structures expressing myogenic markers (myogenin, Troponin T, MF20), though fusion efficiency was markedly reduced compared to myoblasts, indicating limited and likely defective myogenic differentiation capacity. In parallel, adipogenic and osteogenic potentials were strongly impaired, as demonstrated by reduced lipid and calcium deposition, altered expression of FABP4 and leptin. Moreover, DUX4-expressing MSCs displayed pro-fibrotic features, including enhanced collagen III/IV and fibronectin, suggesting impaired extracellular matrix turnover. Together, these findings indicate that DUX4 induces a unique phenotype in MSCs, characterized by impaired differentiation, oxidative stress, partial myogenic reprogramming, and pro-fibrotic activity, contributing to muscle pathology in FSHD.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Beyond Myoblasts: DUX4 Drives Fibrosis and Myogenic Reprogramming in Mesenchymal Stem Cells\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-23 10:45:31\",\"doi\":\"10.21203/rs.3.rs-9094205/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"e60254c3-23b2-4bd0-a142-b375138c3054\",\"owner\":[],\"postedDate\":\"March 23rd, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-25T21:23:25+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-23 10:45:31\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9094205\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9094205\",\"identity\":\"rs-9094205\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}