Transcriptomic Profiling of Muscle-Derived Stromal and Adipocytes Cells Exposed to Suborbital Microgravity

Transcriptomic Profiling of Muscle-Derived Stromal and Adipocytes Cells Exposed to Suborbital Microgravity | 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 Transcriptomic Profiling of Muscle-Derived Stromal and Adipocytes Cells Exposed to Suborbital Microgravity Sergio Perez-Diaz, Jaime Granado Leon, Håkan Rundqvist, Anastasios Damdimopoulos, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7745208/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Spaceflight negatively impacts skeletal muscle mass and function, and current countermeasures fail to completely offset those alterations. Innovative approaches are then needed to understand the mechanisms governing space-induced muscle changes. This study explores short-term microgravity (suborbital flight) effects on transcriptional profile of skeletal muscle mesenchymal cells and intramuscular adipose tissue. We identified a set of molecular factors and biological processes impacted by microgravity in these cell types, warranting further investigation. Biological sciences/Cell biology Biological sciences/Physiology Skeletal muscle Mesenchymal stem cells Intramuscular adipose tissue Suborbital flight Space stressors Figures Figure 1 Figure 2 Figure 3 Figure 4 Main text Space exploration is one of the greatest challenges for humankind, encompassing both technological and biological aspects. Humans are not adapted to extraterrestrial conditions such as the absence of gravity and the intense exposure to cosmic radiation Furthermore, space crews experience chronic stress, which, combined with the space exposome, has a negative impact on human health. The skeletal muscle is one of the most heavily affected physiological systems by the space environment. One month in space results in a 20% decrease in muscle mass and a 30% reduction in muscle strength 1 . The reduction of muscle mass and strength negatively impacts movement, locomotion, and metabolism. Despite the partial positive effect of countermeasures during spaceflight, there is still a need for optimizing standard therapies to counteract the detrimental effects of space on muscle mass and function, which will help preserve crew health and performance, and thus the success of the missions. Changes in adult muscle mass are controlled by the intrinsic molecular processes of muscle fibers 2 . In turn, muscle fibers are strongly modulated by the muscle stroma. The skeletal muscle stroma is a connective tissue composed of a complex extracellular matrix (ECM) and a pool of different cell types, including endothelial cells, hematopoietic cells, fibroblasts, adipocytes, and mesenchymal/stromal stem cells (MSCs). MSCs are pluripotent stem cells that have been studied in the field of muscle development, repair, and disease 3 , due to their capacity to differentiated into different cell types and to their role in the primary functions of muscle 45 . Recent studies have shown that MSCs undergo acute transcriptional changes upon anti muscle atrophy interventions 6 . However, little is known about how the human skeletal muscle stroma responds to microgravity-induced atrophy. Given the limited information on the effects of microgravity/space stressors on these muscle cell types, we examined the transcriptional response of 3D models of skeletal muscle MSCs and intramuscular adipose tissue (IMAT) using the sounding rocket Mapheous-14 as a microgravity analog. Compared with earthbound microgravity simulators that rely on adding directional opposing forces than result in a net 0 g force vector 7 , sounding rockets allow for a short period of real microgravity (~6 minutes). Results obtained employing suborbital rockets can then be used as a valuable reference for the refinement of earthbound simulation studies, as well as hypothesis generators for follow-up low-Earth orbit experiments (e.g. long-term real microgravity aboard the International Space Station). Prior to the suborbital flight, we validated our IMAT and MSCs models. The CD56 - 3D models were either exposed to adipogenic medium (AM) or maintained in growth medium for ten days. We determined fibroblast markers and lipid droplet formation using α-Smooth Muscle Actin (α-SMA) and Perilipin-1 antibodies, respectively. AM-treated models (+AM) increased Perilipin-1, and decreased α-SMA expression compared to controls (-AM) (Figure 1). Transcript levels of ACTA2 (α-SMA) and PLIN1 (Perilipin-1) in +AM models showed significant (p<0.0001) decreases (-1.25-fold-change to -AM) and increases (7.67-fold-change to -AM), respectively. BODIPY™ staining (green) showed consistent lipid accumulation in the AM-treated models (Supplementary Video 1) compared to the controls (Supplementary Video 2). One set of these validated models was loaded aboard the Mapheus-14 sounding rocket, while a second set of both models was kept on the ground as a control. After the suborbital flight, 3D models RNA was extracted, sequenced, and analyzed. Compared to the ground controls, the models of muscle MSCs loaded on board the Mapheaus-14 showed upregulated expression (False Discover Rate; FDR<0.05) of CA3, associated with fatigue and slow fibers 8 ; COL5A2 , previously found upregulated on human analogues of spaceflight 9 and realmicrogravity 10 and SLC5A3 , which has previously been related with myositis 11 . MN1 , TMEM30B and the long non-coding RNA lncZIC1 were also upregulated after microgravity (Figure 2A; Supplementary Table 1). Upregulated Differentially Expressed Genes (DEGs, nominal p-value<0.05) in the microgravity models compared to ground controls, clustered in pathways such as: ECM/basal lamina and focal adhesion/cytoskeleton (e.g. Genes encoding for Collagens (12), laminins (4), NID1 , TNC , FN1 and ITGA11 ); TGF-β signaling (e.g. TGFB2 and TGFBR3 ); fat differentiation (e.g. CEBPA and SREBF1 ); glucose homeostasis (e.g. IGF1 ) and chromatin remodeling (e.g. KDM5A and KDM5B ) (Figure 2B and Supp. Table 2). The expression of PTGDS, which is involved in muscle adaptation to exercise 12 , D AZL , DBP and TLL2 , which is involved in muscle prenatal morphogenesis 13 were found downregulated (FDR<0.05) in the models of muscle MSCs when compared to ground controls (Figure 2A; Supplementary Table 1). The downregulated DEGs (nominal p-value<0.05) were clustered around angiogenesis (e.g. VASH2 , VEGFB and VEGFD ), thin filament proteins, cytokine regulation (e.g. CXCL12 and JUN ), chromatin condensation (e.g. H1-4 , H1-2 , and NCAPH2 ), and glycerophospholipid metabolism (Figure 2C; Supp. Table 2). In the IMAT models, we found 166 DEGs between the microgravity and the ground-control groups. From those, 114 DEGs were upregulated and 52 downregulated. These included 9 long non-coding RNAs (4 up-, 5 down-regulated). CITED2 , which has been related to glucocorticoids 14 , and the adiposity marker Adipsin ( CFD ) 15 were found strongly upregulated after microgravity (Figure 3A; Supplementary Table 3). The functional annotation (nominal p-value<0.05) revealed an upregulation of ribosome biogenesis genes (e.g. Cytoplasmic ( RPL/RPS ) (61) and mitochondrial ( MRPL/MRPS ) (22) ribosomal genes); translation (RNA polymerase II subunits (7); mitochondrial function/redox stress (e.g. NADH ubiquinone oxidoreductase (10), ATP synthase (15), and cytochrome C oxidase (9)); signal transduction (e.g. JUN and FOS ), anabolic metabolism, proteasomal degradation (Proteasome subunits ( PSM ) (8)), and senescence/inflammation (e.g. H2XA , CDKN2A , CXCL8 and IL-32) (Figure 3B; Supp. Table 4). Among the DEGs downregulated by microgravity exposure (FDR<0.05), we found Dystrophin (DMD) and LAMA2, both involved in congenital muscle distrophy 16 . The kynureninase enzyme (KYNU), involved in the biosynthesis of NAD and muscle metabolic homeostasis during exercise 17 , and NEAT1, a long non-coding RNA which controls mitochondrial function (Figure 3A; Supp. Table3), were also downregulated. The downregulated DEGs (nominal p-value<0.05) clustered in pathways involving cell adhesion, cytoskeleton, and vesicle transport. Several pathways were downregulated such as: insulin signaling, PI3K-AKT, JAK-STAT, EGF-EGFR, IL-6, cGMP-PKG, stemness, muscle development, protein ubiquitination, and chromatin remodeling. Downregulation of RAB GTPases (5) and IGFBP5, involved in glucose sensing and, cell survival regulators like MTOR , MDM2 , and PTEN suggest impaired insulin/GLUT4 trafficking and altered survival features on the IMAT models after microgravity exposure, respectively (Figures 3C; Supp. Table 4). The current study is the first to report the transcriptional changes induced by real microgravity, using suborbital flight, on skeletal muscle MSCs and IMAT. Our results show signs of fibrogenic shift of the muscle MSCs exposed to microgravity, as previously seen in adipose tissue-derived MSCs 18 . Among the DEGs induced by microgravity, fibrosis markers such as fibrillar collagen COL5A2 4 showed upregulation. These changes were accompanied by increased expression of enzymes involved in glucose utilization and less condensed chromatin, a phenomenon described in TGF-β-mediated fibrogenesis in other organs 18 . Muscle adaptation to space and other conditions that affect muscle mass require tissue remodeling. Therefore, exposure of the muscle MSCs to space could have induced the phenotypic changes on the MSCs associated to muscle loss of mass and strength 19 . However, the direction of those changes is still a matter of debate 20 . Results from animal analogues of spaceflight 21 and from muscle biopsies from astronauts 22 have shown a decrease in ECM and non-fibrillar collagens, respectively. Interestingly, the same study with astronauts reported a significant increase in the inter-fiber area after spaceflight 22 . Microgravity promote MSC´s adipogenesis 23 and spaceflight analogs, such as bed rest, induced IMAT infiltration 24 , which increases muscle stroma. Primary transcriptional regulators of MSCs adipogenesis, e.g., CEBPA, and adiposity, e.g., Adipsin ( CFD ) appear potentially upregulated in the muscle MSCs and IMAT models exposed to microgravity. This highlights the fibro-adipogenic potential of the human CD56 - muscle cells fraction, and it may explain the increased stromal area observed in astronauts´ muscle 22 . The excessive accumulation of IMAT has been classically associated with poor muscle quality 25 . The suborbital flight seems to benefit the ribosome and mitochondrial function of the IMAT models. Along with a decrease in protein ubiquitination, we found an overexpression of factors clustering in multiple anabolic pathways, including triglyceride biosynthesis, the paramount function of the adipose tissue. The transcriptional changes in IMAT models described here may help explaining the aberrant glucose handling observed in human analogues of spaceflight 26 . Notably, the IMAT models also showed signs of cellular senescence, a feature associated with hypertrophic adipocytes and abnormal glucose metabolism 27 . Currently, there is no consensus about the deleterious consequences of excessive IMAT accumulation. Some authors have argued that the quality of the IMAT has more pathological relevance than quantity 28 . The increase of SAPS factors, deregulation of JAK-STAT and Interleukin-6-mediated pathways observed in the IMAT models subjected to suborbital spaceflight, indicates altered endocrine function, which can certainly affect muscle function. The interpretation of our findings should consider the absence of mechanistic assessments of microgravity-induced DEGs in muscle stroma and IMAT, as well as their specific effects on muscle phenotype. Further studies are warranted to validate and extend these observations. Nonetheless, this study introduces adaptable models of muscle MSCs and IMAT tissue and proposes a set of markers and biological processes that may serve as important contributors to the physiological adaptations of skeletal muscle to spaceflight. Material and Methods Human samples and primary cell cultures Skeletal muscle biopsies were obtained from the vastus lateralis muscle in a cohort of seven healthy volunteers (mean age: 24.8 ± 6.6 years; five males, two female) using the Bergstrom needle technique with suction. Prior to undergoing the procedure, all participants were required to read and sign an informed consent form. All the experiments were approved by the Swedish Ethical Review Authority (#2022-04662-01, with addendums #2023-05072-02 and #2023-05072-02). The tissue (~100 mg) was meticulously minced using a scalpel for up to ten minutes. Then, the homogenates were enzymatically disaggregated in digestion media [2 mg/ml Collagenase D (#11088858001, Sigma-Aldrich), 2 mg/ml Dispase II (#D4693, Sigma-Aldrich), 1% bovine serum albumin (BSA, #A9418, Sigma-Aldrich), 1% penicillin-streptomycin (#15140122, GibcoTM, ThermoFisher) in Dulbecco's modified Eagle medium: nutrient mixture F-12 (DMEM:F-12, #21331020, Gibco™, ThermoFisher)] for 1-hour on a rocking platform at 37°C. The samples were subsequently filtered through 70 µm and 40 µm strainers to remove the debris. The flowthroughs were centrifuged at 300g for five minutes at room temperature. The supernatants were discarded, and the pellets (cells) were subsequently resuspended in a maintenance medium composed of a 1:3 ratio of HAMS F-12 (#11765054, Gibco TM, ThermoFisher), 1:3 ECBM (Endothelial Cell Basal Medium, #MBS652968, MyBioSource), 1:3 Roswell Park Memorial Institute (RPMI, #61870010, Gibco TM, ThermoFisher), 20% Fetal Bovine Serum (FBS, #F9665, Sigma-Aldrich), and 1% Penicillin-Streptomycin. The muscle mononuclear fractions were cultured in vitro at 37°C and 5% CO 2 in a humidified incubator under strict sterility conditions. Cultures at 80-90% confluence were washed with DPBS (#14190144, GibcoTM, ThermoFisher), treated with TrypLE (#12605028, GibcoTM, ThermoFisher), and cryopreserved in FBS 10% DMSO (#D2650, Sigma-Aldrich) at -80°C until further use. Cell sorting Cryovials containing the skeletal muscle mononuclear fraction were rapidly thawed in a pre-warmed water bath and subjected to cell-population sorting. The mononuclear cell fractions from skeletal muscle were incubated with an anti-CD56 antibody (#130-090-955, Miltenyi Biotec) and separated using anti-mouse magnetic beads (#130-048-402, Miltenyi Biotec) in sorting buffer (2 mM EDTA (#E5134, Sigma-Aldrich), 0.5% BSA, 0.1% FBS, and 1% P/S in PBS) according to the manufacturer's instructions. The CD56 + fraction (Myogenic linage) was separated and the CD56- cell fraction (non- myogenic linage/Stromal/Mesenchymal fraction) were counted using a Cell Drop BF (Denovix) and plated at the density of 2500 cells/cm 3 according to the downstream experiments, and kept until the thirteenth to fifteenth passage, moment when the cell lines were discarded. 3D models of human intramuscular stroma and adipose tissue The 3D models were generated by centrifugation (5804, Eppendorf) of 200.000 CD56- cells at 1200 rpm for five minutes in 500µl of maintenance medium [1/3 ECBM, 1/3 HAMS F-12, 1/3 RPMI, 1X Insulin-Transferrin-Selenium-Sodium Pyruvate (ITS-A, #51300044, GibcoTM, ThermoFisher), 1/2000 Fatty Acid Supplement (#F7050, Sigma-Aldrich), 0.5mg/mL BSA 0.05%, Dexamethasone (#D4902, Sigma-Aldrich), 0.5mg/mL Fetuin (#F53385, Sigma-Aldrich), 4ng/mL basic Fibroblast Growth Factor (bFGF, #NBP2-34921, NovusBio), 4ng/mL Fibroblast Growth Factor 4 (FGF4, #235-F4, R&D Systems), 4ng/mL Epidermal Growth Factor (EGF, #236-BPF, R&D Systems), 4ng/mL Insulin Growth Factor 1 (IGF1, #100-11, PeproTech), 1X L-Glutamine (#35050038, ThermoFisher) and 1% Penicillin-Streptomycin (P/S)] using falcon round bottom tubes (#60819-310, VWR) 29 . The cultures were kept in maintenance media for 5 days, which provided enough time for the cultures to assemble in an organoid-like 3D model. Ten days before exposure to microgravity, the cultures were exposed to the adipogenic medium [1/3 ECBM, 1/3 HAMS F-12, 1/3 RPMI, 10ug/ml insulin (#11061-68-0, Sigma-Aldrich), 5ug/ml Rosiglitazone (#122320-73-4, Cayman chemical), 1X L-Glutamine, 0,1% BSA and 1% P/S] 30 to induce the differentiation of the mesenchymal stem cells into adipocyte and generate the intramuscular adipose tissue in vitro or kept in maintenance media (Fibroblasts/stroma). After treatment, the models were processed for histological analysis or exposed to microgravity/ground-control and subsequently used to extract the RNA. Immunofluorescence The models were washed twice with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) (#11586711, ThermoFisher) for two hours at 4°C. The PFA was removed, and the samples were washed with PBS, before being treated with 30% sucrose overnight at 4ºC. The 3D models were embedded into OCT (#45830, Histolab), frozen in cold liquid nitrogen isopentane (VWR, #24872.298) and stored at -80°C until sectioning. Sections of 7µm were obtained using the CryoStar™ NX50 Cryostat (ThermoFisher). The entire 3D models and 3D models´ sections were permeabilized using a solution of 0.05% Triton-X (#T9284, Sigma-Aldrich) in PBS for 10 minutes, washed and blocked with 10% BSA in PBS for 30 minutes. After blocking, samples were washed again with PBS and incubated overnight at 4ºC with a solution of 0.01% Triton-X and 1% BSA in PBS containing the primary antibodies [Mouse anti α-Smooth Muscle Actin, 1:100 (#sc-53142, Santa Cruz Biotechnologies) and Rabit anti Perlipin-1, 1:200 (#ab3526, Abcam)]. Samples were washed with 0.02 % Tween-20 (#P7949, Sigma-Aldrich) in PBS (TPBS) and incubated with the secondary antibody [Goat anti Mouse Alexa Flour 568 (#A11004, ThermoFisher) and Goat anti Rabbit Alexa Flour 488 (#A11008, ThermoFisher)] or BODIPY ™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) 1/1000 to stain the neutral lipids in PBS for 30 minutes at 37°C. The samples were washed three times with TPBS and mounted using ProLong™ Gold Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI) (#P36931, ThermoFisher). The images were captured using Zeiss AXIO Observer Z1 inverted microscope and Zeiss Zen pro software (Carl Zeiss). Videos were recorded with a Nikon Confocal A1R+ (Nikon) microscopy and Nikon Imaging Software (NIS) Elements Advanced Research Software version 5.30.02 (Nikon, Tokyo, Japan). The fluorescence signal of the immunoassayed proteins (α-SMA, Laminin, MyH1) in the spheroids was calculated as the percentage of area occupied by the signal of the target protein per total area of the image (in pixels) and normalized by the area of the spheroid (DAPI signal) using a macro for Image J software 31 developed and kindly provided by Benedicte Chazaud, Institute NeuroMyoGène, Lyon, France. Suborbital flight on board of a microgravitational vehicle. The 3D models of MSCs and IMAT were transferred into 2ml cryotubes containing their respective media. A custom-build cartridge (Swedish Space Corporation, Sweden) was used to secure the 3D models into the Mapheous 14 sounding rocket shared module, launched from Esrange Space Center (Kiruna, Sweden) on the 27th of February of 2024. Six IMAT and six MSC 3D models were subjected to spaceflight. The same number of 3D models remained in the lab of Esrange Space Center as ground controls. Following the suborbital flight, the samples were recovered (within 2 hours after landing), all 3D models were promptly washed in PBS, resuspended in TRIzol to preserve the RNA, froze in dry ice, and shipped in dry ice to the laboratories of the Space & Environmental Physiology group at Karolinska Institutet for transcriptional analysis (Figure 4). RNA extraction and sequencing and transcriptional analysis The 3D models’ total RNA was extracted following the column-based protocol previously described 29 . SMART-Seq libraries were synthesized from total RNA using SMART-Seq® v4 Ultra® Low Input RNA Kit (TaKaRa Bio USA, Mountain View, CA, USA) according to the manufacturer’s instructions. The first-strand cDNAs were synthesized from the total RNA using nontemplated nucleotides added by the SMARTScribe™ Reverse Transcriptase, then, the double-strand cDNAs were synthesized by template switching. PCR was directly performed and samples purified. Quality control of cDNAs were performed on Agilent Tapestation according to the manufacturer’s instructions. 1 ng of cDNA was used for the tagmentation reaction. During tagmentation, samples are fragmented and tagged with adapters, and a PCR is done to amplify the tagged DNA and sequencing indexes are added. The indexed libraries were purified, normalised and combined. The pool was sequenced on the Illumina Nextseq 2000 on a P2 100 flowcell sequencing run, generating 2x60 bp paired end reads. Bcl files were converted and demultiplexed to fastq using the bcl2fastq program. STAR v2.7.11b was used to index the human reference genome (hg38/GRCh38) and align the resulting fastq files. Mapped reads were then counted in annotated exons using featureCounts. The gene annotations (Homo_sapiens.GRCh38.113.gtf) and reference genome were obtained from Ensembl. The count table from featureCounts was imported into R/Bioconductor and differential gene expression was performed using the EdgeRpackage and its general linear model’s pipeline. For the gene expression analysis genes were filtered using the filterByExpr function and normalized using TMM normalization. Genes with an FDR adjusted p value <0.05 were termed significantly regulated. GO and pathway analysis was conducted using the goana, kegga and camera functions from the limma packagem, while Wikipathways were retrieved using the rWikiPathways R library. Statistical analysis Statistical analyses were conducted using unpaired t-tests between the 3D models exposed to for ten days to adipogenic medium (+AM) and control (-AM; kept in maintenance medium). Outliers were detected and removed using the Tukey test. Figure 1 statistical procedures and graphs were carried out in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A significance threshold of 5% (p-value < 0.05) was used. Declarations Authors contribution SP-D: experimental design, laboratory work and methods, data analysis and visualization, interpretation of the results, funding, manuscript drafting and review. JGL: laboratory work and methods, manuscript review. HR: laboratory work and methods, data visualization, manuscript review. AD: transcriptional methods and analysis, manuscript review. TG: interpretation of the results, manuscript review. RF-G: experimental design, interpretation of the results, funding, manuscript drafting and review, project coordination. Ethical The experiments included in this study were performed according to principles of the declaration of Helsinki and approved by the Swedish Ethical Review Authority (#2022-04662-01, with addendums #2023-05072-02 and #2023-05072-02). Samples were obtained with written informed consent. The study is registered in ClinicalTrials.gov, with registration number NCT05801185. Consent for publication Not applicable. Funding This project has been funded by Gösta Fraenckels stiftelse, with grants to SPD (#2023-01797 and #2024-02295) and RFG (#2023-01774 and #2024-02297). RFG is supported by a career grant from the Swedish National Space Agency (#2021-00159). SPD is supported by IBSA foundation fellowship 2024. Availability of data and materials The datasets and material analyzed in this study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Acknowledgement The authors are grateful to for the invaluable technical assistance made by BEA (Bioinformatics and Expression Analysis) core facility at Novum, Karolinska Institutet in Huddinge (Sweden). Likewise, the authors thank the Swedish Space corporation (SSC) for their helpful operational contribution to the study, in particular Stefan Krämer, Gunnar Florin, and Marton Galbacs. The authors declare that they have not used Artificial Intelligence in this study. 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Supplementary Files Supp.Table1.xlsx Supp.Table2.xlsx Supp.Table3.xlsx Supp.Table4.xlsx Supp.video1.pptx Supp.video2.pptx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 20 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 04 May, 2026 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 07 Oct, 2025 First submitted to journal 29 Sep, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7745208","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":529540018,"identity":"0fb221b0-f1a4-4a03-936a-bcb3a3bbb033","order_by":0,"name":"Sergio Perez-Diaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYLCCBDiLTYKBgb2N4QCJWniOEaEFAdiAWCINvxr59t7HLx5UMOTxSx8+/IGhzEJefuazxAMMf2xwajE4c9zMIuEMQ7FkX1qCAcM5CcPG2WkHDjC24bbKQCKNzSCxjSFxwxkegwTGNgnGZun0hgOMDYdxO2z+M4iW/Wf4PwANl7BvkzzeAHTYf9yeucHG/ABsCw8PYwNQS2KPBNuBAwxsB/D4JY2NIeGMRLHEGTZjhoRzEskzeNISDiS2JeN2WPsx5o8/Kmzy+HuYH3/4UFZnO7/9mPGHD3/scDsMEn0SCWBmAkwsAbtSGGD+QFjNKBgFo2AUjGgAADmOUdwaSItlAAAAAElFTkSuQmCC","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Perez-Diaz","suffix":""},{"id":529540019,"identity":"505948ab-e16b-4385-83e7-17f4eeb7fb64","order_by":1,"name":"Jaime Granado Leon","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Jaime","middleName":"Granado","lastName":"Leon","suffix":""},{"id":529540021,"identity":"41236db8-c921-4c08-ac15-d1e4cf0f6f1d","order_by":2,"name":"Håkan Rundqvist","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Håkan","middleName":"","lastName":"Rundqvist","suffix":""},{"id":529540022,"identity":"d7025bd4-b89a-454a-a609-1010ddb3fe33","order_by":3,"name":"Anastasios Damdimopoulos","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anastasios","middleName":"","lastName":"Damdimopoulos","suffix":""},{"id":529540023,"identity":"3f8a0df4-93f7-4c10-906e-3d41a011f7ee","order_by":4,"name":"Thomas Gustafsson","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Gustafsson","suffix":""},{"id":529540024,"identity":"9348b965-92ba-43ce-9df0-d0c6966087a6","order_by":5,"name":"Rodrigo Fernandez-Gonzalo","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Fernandez-Gonzalo","suffix":""}],"badges":[],"createdAt":"2025-09-29 20:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7745208/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7745208/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97993802,"identity":"13534fd1-0cf6-4ef4-a2a5-7d88aaab7b8f","added_by":"auto","created_at":"2025-12-11 14:55:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1488491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTop: Immunofluorescence of skeletal muscle CD56\u003csup\u003e-\u003c/sup\u003e 3D models (Model 2 and 3) exposed for ten days to adipogenic medium (+AM) and control (-AM; kept in maintenance medium; Model 1 and 2). Sections were stained for α-Smooth Muscle Actin (α-SMA, red) and Perlipin-1 (green). Nuclei are stained with DAPI (blue). Scale bars: 100 µm.\u003c/p\u003e\n\u003cp\u003eBotton: Signal quantification of α-SMA and Perlipin-1. The results are presented as scatter plot with bar of the Mean ± Standard deviation. Dots are independent 3D model values (n= 5).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/1d2749863e98b06b4639f35a.jpg"},{"id":97993799,"identity":"e564b2d3-7449-42b6-b551-004bddc43063","added_by":"auto","created_at":"2025-12-11 14:55:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1371808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed sequences and biological processes of skeletal muscle stroma 3D models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Volcano plot illustrates the differentially expressed genes (DEGs) (p-value\u0026lt;0.05, grey color. Empty dots: False Discovery Rate (FDR) \u0026lt; 0.05) in the 3D models of skeletal muscle stroma subjected to suborbital flight (n=4) compared to ground control (n=4).\u003c/p\u003e\n\u003cp\u003eB) Gene ontology of up-regulated biological processes (DAVID; red color) and pathways (KEGG; green color, WikiPathways; blue color) enriched in the 3D models of skeletal muscle stroma subjected to suborbital flight (n=4) compared to ground control (n=4) with p-value \u0026lt; 0.05. The size of the circle is indicative of the number of annotated genes within a group included in a particular gene ontology or pathway.\u003c/p\u003e\n\u003cp\u003eC) Gene ontology of down-regulated biological processes (DAVID; red color) and pathways (KEGG; green color, WikiPathways; blue color) enriched in the 3D models of skeletal muscle stroma subjected to suborbital flight (n=4) compared to ground control (n=4) with p-value \u0026lt; 0.05. The size of the circle is indicative of the number of annotated genes within a group included in a particular gene ontology or pathway.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/a8e4b7cb9ba9450979010b1b.jpg"},{"id":97993804,"identity":"22648551-d9d1-4702-b2c3-cd0c2052a5f6","added_by":"auto","created_at":"2025-12-11 14:55:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1807260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed sequences and biological processes of intramuscular adipose tissue 3D models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Volcano plot illustrates the differentially expressed gene (DEGs) (p-value\u0026lt;0.05, grey color. Empty dots: Selected DES with False Discovery Rate (FDR) \u0026lt; 0.05) in the 3D models of intramuscular adipose tissue subjected to suborbital flight (n=4) compared to ground control (n=4).\u003c/p\u003e\n\u003cp\u003eB) Gene ontology of up-regulated biological processes (DAVID; red color) and pathways (KEGG; green color, WikiPathways; blue color) enriched in the 3D models of intramuscular adipose tissue subjected to suborbital flight (n=4) compared to ground control (n=4) with p-value \u0026lt; 0.05. The size of the circle is indicative of the number of annotated genes within a group included in a particular gene ontology or pathway.\u003c/p\u003e\n\u003cp\u003eC) Gene ontology of down-regulated biological processes (DAVID; red color) and pathways (KEGG; green color, WikiPathways; blue color) enriched in the 3D models of intramuscular adipose tissue subjected to suborbital flight (n=4) compared to ground control (n=4) with p-value \u0026lt; 0.05. The size of the circle is indicative of the number of annotated genes within a group included in a particular gene ontology or pathway.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/6f3f6d9450b31502595d514d.jpg"},{"id":97993803,"identity":"a6f32adf-b400-40f3-8956-5b9c5667ddc3","added_by":"auto","created_at":"2025-12-11 14:55:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic simplification of the experimental design.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human samples of skeletal muscle were digested and the non-myogenic population sorted. Once the primary cultures reached the number of progenitor cells needed, the 3D models were established and differentiated using the different medium formulation. The models were then transferred into an aseptic cuvette, integrated into a particular cartridge, placed in the sounding rocket, and subjected to a suborbital flight. Equally generated models were treated similarly but kept as ground control. After suborbital flight, the models were immediately recovered and transferred to TRizol. Finally, the RNA was isolated, sequenced, and analyzed.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/0f9b030222f8e0eb2b3c2f94.jpg"},{"id":98797625,"identity":"e5be2635-7391-43ef-88de-88d772c668ea","added_by":"auto","created_at":"2025-12-22 13:36:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5353116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/33a41280-580e-4106-898f-efddf6a79764.pdf"},{"id":97900364,"identity":"ecadf8c6-847b-43ac-a6e1-0fa8bcc3a3aa","added_by":"auto","created_at":"2025-12-10 15:45:25","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33391,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/ce81e95c8e9a10fd7545281c.xlsx"},{"id":97860915,"identity":"81f616e0-4782-441d-ba1f-340c5e20e505","added_by":"auto","created_at":"2025-12-10 08:47:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19500,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/3c4e5e697ffeccf723a98ade.xlsx"},{"id":97899247,"identity":"0508a7e3-df6a-469c-899d-d4e0bb167180","added_by":"auto","created_at":"2025-12-10 15:42:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":103388,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/f64a5640b73dcc1bf98e464f.xlsx"},{"id":97860918,"identity":"a6c4b66c-66c6-42d3-bab1-9bde3f1cc1f6","added_by":"auto","created_at":"2025-12-10 08:47:16","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":30672,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/6889e4189014aa3c69c59675.xlsx"},{"id":97900128,"identity":"e738a6ef-4c9e-4157-80c9-ee11f0b70f75","added_by":"auto","created_at":"2025-12-10 15:45:14","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6870270,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.video1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/c57c9111dd9d5ebbba7603d0.pptx"},{"id":97860922,"identity":"4e53a894-fd31-4a91-898e-04ece4eb4907","added_by":"auto","created_at":"2025-12-10 08:47:16","extension":"pptx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":5948984,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.video2.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7745208/v1/e8b7769f89388547e0dc5872.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptomic Profiling of Muscle-Derived Stromal and Adipocytes Cells Exposed to Suborbital Microgravity","fulltext":[{"header":"Main text ","content":"\u003cp\u003eSpace exploration is one of the greatest challenges for humankind, encompassing both technological and biological aspects. Humans are not adapted to extraterrestrial conditions such as the absence of gravity and the intense exposure to cosmic radiation Furthermore, space crews experience chronic stress, which, combined with the space exposome, has a negative impact on human health. The skeletal muscle is one of the most heavily affected physiological systems by the space environment. One month in space results in a 20% decrease in muscle mass and a 30% reduction in muscle strength\u003csup\u003e1\u003c/sup\u003e. The reduction of muscle mass and strength negatively impacts movement, locomotion, and metabolism. Despite the partial positive effect of countermeasures during spaceflight, there is still a need for optimizing standard therapies to counteract the detrimental effects of space on muscle mass and function, which will help preserve crew health and performance, and thus the success of the missions.\u003c/p\u003e\n\u003cp\u003eChanges in adult muscle mass are controlled by the intrinsic molecular processes of muscle fibers\u003csup\u003e2\u003c/sup\u003e. In turn, muscle fibers are strongly modulated by the muscle stroma. The skeletal muscle stroma is a connective tissue composed of a complex extracellular matrix (ECM) and a pool of different cell types, including endothelial cells, hematopoietic cells, fibroblasts, adipocytes, and mesenchymal/stromal stem cells (MSCs). MSCs are pluripotent stem cells that have been studied in the field of muscle development, repair, and disease\u003csup\u003e3\u003c/sup\u003e, due to their capacity to differentiated into different cell types and to their role in the primary functions of muscle\u003csup\u003e45\u003c/sup\u003e. Recent studies have shown that MSCs undergo acute transcriptional changes upon anti muscle atrophy interventions\u003csup\u003e6\u003c/sup\u003e. However, little is known about how the human skeletal muscle stroma responds to microgravity-induced atrophy. Given the limited information on the effects of microgravity/space stressors on these muscle cell types, we examined the transcriptional response of 3D models of skeletal muscle MSCs and intramuscular adipose tissue (IMAT) using the sounding rocket Mapheous-14 as a microgravity analog. Compared with earthbound microgravity simulators that rely on adding directional opposing forces than result in a net 0 g force vector\u003csup\u003e\u0026nbsp;7\u003c/sup\u003e, sounding rockets allow for a short period of real microgravity (~6 minutes). Results obtained employing suborbital rockets can then be used as a valuable reference for the refinement of earthbound simulation studies, as well as hypothesis generators for follow-up low-Earth orbit experiments (e.g. long-term real microgravity aboard the International Space Station).\u003c/p\u003e\n\u003cp\u003ePrior to the suborbital flight, we validated our IMAT and MSCs models. The CD56\u003csup\u003e-\u003c/sup\u003e 3D models were either exposed to adipogenic medium (AM) or maintained in growth medium for ten days. We determined fibroblast markers and lipid droplet formation using α-Smooth Muscle Actin (α-SMA) and Perilipin-1 antibodies, respectively. AM-treated models (+AM) increased Perilipin-1, and decreased α-SMA expression compared to controls (-AM) (Figure 1). Transcript levels of \u003cem\u003eACTA2\u003c/em\u003e (α-SMA) and \u003cem\u003ePLIN1\u003c/em\u003e (Perilipin-1) in +AM models showed significant (p\u0026lt;0.0001) decreases (-1.25-fold-change to -AM) and increases (7.67-fold-change to -AM), respectively. BODIPY™ staining (green) showed consistent lipid accumulation in the AM-treated models (Supplementary Video 1) compared to the controls (Supplementary Video 2). One set of these validated models was loaded aboard the Mapheus-14 sounding rocket, while a second set of both models was kept on the ground as a control.\u003c/p\u003e\n\u003cp\u003eAfter the suborbital flight, 3D models RNA was extracted, sequenced, and analyzed. Compared to the ground controls, the models of muscle MSCs loaded on board the Mapheaus-14 showed upregulated expression (False Discover Rate; FDR\u0026lt;0.05) of \u003cem\u003eCA3,\u003c/em\u003e associated with fatigue and slow fibers\u003csup\u003e8\u003c/sup\u003e; \u003cem\u003eCOL5A2\u003c/em\u003e, previously found upregulated on human analogues of spaceflight\u003csup\u003e9\u0026nbsp;\u003c/sup\u003eand realmicrogravity\u003csup\u003e10\u003c/sup\u003e and \u003cem\u003eSLC5A3\u003c/em\u003e, which has previously been related with myositis\u003csup\u003e11\u003c/sup\u003e. \u003cem\u003eMN1\u003c/em\u003e, \u003cem\u003eTMEM30B\u003c/em\u003e and\u0026nbsp;the long non-coding RNA lncZIC1 were also upregulated after microgravity (Figure 2A; Supplementary Table 1). Upregulated Differentially Expressed Genes (DEGs, nominal p-value\u0026lt;0.05) in the microgravity models compared to ground controls, clustered in pathways such as: ECM/basal lamina \u0026nbsp;and focal adhesion/cytoskeleton (e.g. Genes encoding for \u0026nbsp;Collagens (12), laminins (4), \u003cem\u003eNID1\u003c/em\u003e, \u003cem\u003eTNC\u003c/em\u003e, \u003cem\u003eFN1\u003c/em\u003e and \u003cem\u003eITGA11\u003c/em\u003e); TGF-β signaling (e.g. \u003cem\u003eTGFB2\u003c/em\u003e and \u003cem\u003eTGFBR3\u003c/em\u003e); fat differentiation (e.g. \u003cem\u003eCEBPA\u003c/em\u003e and \u003cem\u003eSREBF1\u003c/em\u003e); glucose homeostasis (e.g. \u003cem\u003eIGF1\u003c/em\u003e) and chromatin remodeling (e.g. \u003cem\u003eKDM5A\u003c/em\u003e and \u003cem\u003eKDM5B\u003c/em\u003e) (Figure 2B and Supp. Table 2).\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003ePTGDS,\u003c/em\u003e which is involved in muscle adaptation to exercise\u003csup\u003e12\u003c/sup\u003e, D\u003cem\u003eAZL\u003c/em\u003e, \u003cem\u003eDBP\u003c/em\u003e and \u003cem\u003eTLL2\u003c/em\u003e, which is involved in muscle prenatal morphogenesis\u003csup\u003e13\u003c/sup\u003e were found downregulated (FDR\u0026lt;0.05) in the models of muscle MSCs when compared to ground controls (Figure 2A; Supplementary Table 1). The downregulated DEGs (nominal p-value\u0026lt;0.05) were clustered around angiogenesis (e.g. \u003cem\u003eVASH2\u003c/em\u003e, \u003cem\u003eVEGFB\u003c/em\u003e and \u003cem\u003eVEGFD\u003c/em\u003e), thin filament proteins, cytokine regulation (e.g. \u003cem\u003eCXCL12\u003c/em\u003e and \u003cem\u003eJUN\u003c/em\u003e), chromatin condensation (e.g. \u003cem\u003eH1-4\u003c/em\u003e, \u003cem\u003eH1-2\u003c/em\u003e, and \u003cem\u003eNCAPH2\u003c/em\u003e), and glycerophospholipid metabolism (Figure 2C; Supp. Table 2).\u003c/p\u003e\n\u003cp\u003eIn the IMAT models, we found 166 DEGs between the microgravity and the ground-control groups. From those, 114 DEGs were upregulated and 52 downregulated. These included 9 long non-coding RNAs (4 up-, 5 down-regulated). \u003cem\u003eCITED2\u003c/em\u003e, which has been related to glucocorticoids\u003csup\u003e14\u003c/sup\u003e, and the adiposity marker Adipsin (\u003cem\u003eCFD\u003c/em\u003e)\u003csup\u003e15\u003c/sup\u003ewere found strongly upregulated after microgravity (Figure 3A; Supplementary Table 3). The functional annotation (nominal p-value\u0026lt;0.05) revealed an upregulation of ribosome biogenesis genes (e.g. Cytoplasmic (\u003cem\u003eRPL/RPS\u003c/em\u003e) (61) and mitochondrial (\u003cem\u003eMRPL/MRPS\u003c/em\u003e) (22) ribosomal genes); translation (RNA polymerase II subunits (7); mitochondrial function/redox stress (e.g. NADH ubiquinone oxidoreductase (10), ATP synthase (15), and cytochrome C oxidase (9)); signal transduction (e.g. \u003cem\u003eJUN\u003c/em\u003e and \u003cem\u003eFOS\u003c/em\u003e), anabolic metabolism, proteasomal degradation (Proteasome subunits (\u003cem\u003ePSM\u003c/em\u003e) (8)), and senescence/inflammation (e.g. \u003cem\u003eH2XA\u003c/em\u003e, \u003cem\u003eCDKN2A\u003c/em\u003e, \u003cem\u003eCXCL8\u003c/em\u003e and\u003cem\u003e\u0026nbsp;IL-32)\u003c/em\u003e (Figure 3B; Supp. Table 4).\u003c/p\u003e\n\u003cp\u003eAmong the DEGs downregulated by microgravity exposure (FDR\u0026lt;0.05), we found Dystrophin (DMD) and LAMA2, both involved in congenital muscle distrophy\u003csup\u003e16\u003c/sup\u003e. The kynureninase enzyme (KYNU), involved in the biosynthesis of NAD and muscle metabolic homeostasis during exercise\u003csup\u003e17\u003c/sup\u003e, and NEAT1, a long non-coding RNA which controls mitochondrial function (Figure 3A; Supp. Table3), were also downregulated. The downregulated DEGs (nominal p-value\u0026lt;0.05) clustered in pathways involving cell adhesion, cytoskeleton, and vesicle transport. Several pathways were downregulated such as: insulin signaling, PI3K-AKT, JAK-STAT, EGF-EGFR, IL-6, cGMP-PKG, stemness, muscle development, protein ubiquitination, and chromatin remodeling. Downregulation of RAB GTPases (5) and IGFBP5, involved in glucose sensing and, cell survival regulators like \u003cem\u003eMTOR\u003c/em\u003e, \u003cem\u003eMDM2\u003c/em\u003e, and \u003cem\u003ePTEN\u003c/em\u003e suggest impaired insulin/GLUT4 trafficking and altered survival features on the IMAT models after microgravity exposure, respectively (Figures 3C; Supp. Table 4).\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study is the first to report the transcriptional changes induced by real microgravity, using suborbital flight, on skeletal muscle MSCs and IMAT. Our results show signs of fibrogenic shift of the muscle MSCs exposed to microgravity, as previously seen in adipose tissue-derived MSCs\u003csup\u003e18\u003c/sup\u003e. Among the DEGs induced by microgravity, fibrosis markers such as fibrillar collagen \u003cem\u003eCOL5A2\u003c/em\u003e\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eshowed upregulation. These changes were accompanied by increased expression of enzymes involved in glucose utilization and less condensed chromatin, a phenomenon described in TGF-β-mediated fibrogenesis in other organs\u003csup\u003e18\u003c/sup\u003e. Muscle adaptation to space and other conditions that affect muscle mass require tissue remodeling. Therefore, exposure of the muscle MSCs to space could have induced the phenotypic changes on the MSCs associated to muscle loss of mass and strength\u003csup\u003e19\u003c/sup\u003e. However, the direction of those changes is still a matter of debate\u003csup\u003e20\u003c/sup\u003e. Results from animal analogues of spaceflight\u003csup\u003e21\u003c/sup\u003e and from muscle biopsies from astronauts\u003csup\u003e22\u003c/sup\u003e\u0026nbsp; \u0026nbsp;have shown a decrease in ECM and non-fibrillar collagens, respectively. Interestingly, the same study with astronauts reported a significant increase in the inter-fiber area after spaceflight\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMicrogravity promote MSC´s adipogenesis\u003csup\u003e23\u003c/sup\u003e and spaceflight analogs, such as bed rest, induced IMAT infiltration\u003csup\u003e24\u003c/sup\u003e, which increases muscle stroma. Primary transcriptional regulators of MSCs adipogenesis, e.g., \u003cem\u003eCEBPA,\u003c/em\u003e and adiposity, e.g., Adipsin (\u003cem\u003eCFD\u003c/em\u003e) appear potentially upregulated in the muscle MSCs and IMAT models exposed to microgravity. This highlights the fibro-adipogenic potential of the human CD56\u003csup\u003e-\u003c/sup\u003e muscle cells fraction, and it may explain the increased stromal area observed in astronauts´ muscle\u003csup\u003e22\u003c/sup\u003e. The excessive accumulation of IMAT has been classically associated with poor muscle quality\u003csup\u003e25\u003c/sup\u003e. The suborbital flight seems to benefit the ribosome and mitochondrial function of the IMAT models. Along with a decrease in protein ubiquitination, we found an overexpression of factors clustering in multiple anabolic pathways, including triglyceride biosynthesis, the paramount function of the adipose tissue. The transcriptional changes in IMAT models described here may help explaining the aberrant glucose handling observed in human analogues of spaceflight\u003csup\u003e26\u003c/sup\u003e. Notably, the IMAT models also showed signs of cellular senescence, a feature associated with hypertrophic adipocytes and abnormal glucose metabolism\u003csup\u003e27\u003c/sup\u003e. Currently, there is no consensus about the deleterious consequences of excessive IMAT accumulation. Some authors have argued that the quality of the IMAT has more pathological relevance than quantity\u003csup\u003e28\u003c/sup\u003e. The increase of SAPS factors, deregulation of JAK-STAT and Interleukin-6-mediated pathways observed in the IMAT models subjected to suborbital spaceflight, indicates altered endocrine function, which can certainly affect muscle function.\u003c/p\u003e\n\u003cp\u003eThe interpretation of our findings should consider the absence of mechanistic assessments of microgravity-induced DEGs in muscle stroma and IMAT, as well as their specific effects on muscle phenotype. Further studies are warranted to validate and extend these observations. Nonetheless, this study introduces adaptable models of muscle MSCs and IMAT tissue and proposes a set of markers and biological processes that may serve as important contributors to the physiological adaptations of skeletal muscle to spaceflight.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cem\u003eHuman samples and primary cell cultures\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSkeletal muscle biopsies were obtained from the vastus lateralis muscle in a cohort of seven healthy volunteers (mean age: 24.8 ± 6.6 years; five males, two female) using the Bergstrom needle technique with suction. Prior to undergoing the procedure, all participants were required to read and sign an informed consent form. All the experiments were approved by the Swedish Ethical Review Authority (#2022-04662-01, with addendums #2023-05072-02 and #2023-05072-02). The tissue (~100 mg) was meticulously minced using a scalpel for up to ten minutes. Then, the homogenates were enzymatically disaggregated in digestion media [2 mg/ml Collagenase D (#11088858001, Sigma-Aldrich), 2 mg/ml Dispase II (#D4693, Sigma-Aldrich), 1% bovine serum albumin (BSA, #A9418, Sigma-Aldrich), 1% penicillin-streptomycin (#15140122, GibcoTM, ThermoFisher) in Dulbecco's modified Eagle medium: nutrient mixture F-12 (DMEM:F-12, #21331020, Gibco™, ThermoFisher)] for 1-hour on a rocking platform at 37°C. The samples were subsequently filtered through 70 µm and 40 µm strainers to remove the debris. The flowthroughs were centrifuged at 300g for five minutes at room temperature. The supernatants were discarded, and the pellets (cells) were subsequently resuspended in a maintenance medium composed of a 1:3 ratio of HAMS F-12 (#11765054, Gibco TM, ThermoFisher), 1:3 ECBM (Endothelial Cell Basal Medium, #MBS652968, MyBioSource), 1:3 Roswell Park Memorial Institute (RPMI, #61870010, Gibco TM, ThermoFisher), 20% Fetal Bovine Serum (FBS, #F9665, Sigma-Aldrich), and 1% Penicillin-Streptomycin. The muscle mononuclear fractions were cultured in vitro at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator under strict sterility conditions. Cultures at 80-90% confluence were washed with DPBS (#14190144, GibcoTM, ThermoFisher), treated with TrypLE (#12605028, GibcoTM, ThermoFisher), and cryopreserved in FBS 10% DMSO (#D2650, Sigma-Aldrich) at -80°C until further use.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell sorting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCryovials containing the skeletal muscle mononuclear fraction were rapidly thawed in a pre-warmed water bath and subjected to cell-population sorting. The mononuclear cell fractions from skeletal muscle were incubated with an anti-CD56 antibody (#130-090-955, Miltenyi Biotec) and separated using anti-mouse magnetic beads (#130-048-402, Miltenyi Biotec) in sorting buffer (2 mM EDTA (#E5134, Sigma-Aldrich), 0.5% BSA, 0.1% FBS, and 1% P/S in PBS) according to the manufacturer's instructions. The CD56\u003csup\u003e+\u0026nbsp;\u003c/sup\u003efraction (Myogenic linage) was separated and the CD56- cell fraction (non- myogenic linage/Stromal/Mesenchymal fraction) were counted using a Cell Drop BF (Denovix) and plated at the density of 2500 cells/cm\u003csup\u003e3\u003c/sup\u003e according to the downstream experiments, and kept until the thirteenth to fifteenth passage, moment when the cell lines were discarded.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D models of human intramuscular stroma and adipose tissue\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D models were generated by centrifugation (5804, Eppendorf) of 200.000 CD56- cells at 1200 rpm for five minutes in 500µl of maintenance medium [1/3 ECBM, 1/3 HAMS F-12, 1/3 RPMI, 1X Insulin-Transferrin-Selenium-Sodium Pyruvate (ITS-A, #51300044, GibcoTM, ThermoFisher), 1/2000 Fatty Acid Supplement (#F7050, Sigma-Aldrich), 0.5mg/mL BSA 0.05%, Dexamethasone (#D4902, Sigma-Aldrich), 0.5mg/mL Fetuin (#F53385, Sigma-Aldrich), 4ng/mL basic Fibroblast Growth Factor (bFGF, #NBP2-34921, NovusBio), 4ng/mL Fibroblast Growth Factor 4 (FGF4, #235-F4, R\u0026amp;D Systems), 4ng/mL Epidermal Growth Factor (EGF, #236-BPF, R\u0026amp;D Systems), 4ng/mL Insulin Growth Factor 1 (IGF1, #100-11, PeproTech), 1X L-Glutamine (#35050038, ThermoFisher) and 1% Penicillin-Streptomycin (P/S)] using falcon round bottom tubes (#60819-310, VWR)\u003csup\u003e29\u003c/sup\u003e. The cultures were kept in maintenance media for 5 days, which provided enough time for the cultures to assemble in an organoid-like 3D model. Ten days before exposure to microgravity, the cultures were exposed to the adipogenic medium [1/3 ECBM, 1/3 HAMS F-12, 1/3 RPMI, 10ug/ml insulin (#11061-68-0, Sigma-Aldrich), 5ug/ml Rosiglitazone (#122320-73-4, Cayman chemical), 1X L-Glutamine, 0,1% BSA and 1% P/S]\u003csup\u003e30\u003c/sup\u003e\u0026nbsp; \u0026nbsp;to induce the differentiation of the mesenchymal stem cells into adipocyte and generate the intramuscular adipose tissue in vitro or kept in maintenance media (Fibroblasts/stroma). After treatment, the models were processed for histological analysis or exposed to microgravity/ground-control and subsequently used to extract the RNA.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunofluorescence\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe models were washed twice with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) (#11586711, ThermoFisher) for two hours at 4°C. The PFA was removed, and the samples were washed with PBS, before being treated with 30% sucrose overnight at 4ºC. The 3D models were embedded into OCT (#45830, Histolab), frozen in cold liquid nitrogen isopentane (VWR, #24872.298) and stored at -80°C until sectioning. Sections of 7µm were obtained using the CryoStar™ NX50 Cryostat (ThermoFisher). The entire 3D models and 3D models´ sections\u0026nbsp;were permeabilized using a solution of 0.05% Triton-X (#T9284, Sigma-Aldrich) in PBS for 10 minutes, washed and blocked with 10% BSA in PBS for 30 minutes. After blocking, samples were washed again with PBS and incubated overnight at 4ºC with a solution of 0.01% Triton-X and 1% BSA in PBS containing the primary antibodies [Mouse anti α-Smooth Muscle Actin, 1:100 (#sc-53142, Santa Cruz Biotechnologies) and Rabit anti Perlipin-1, 1:200 (#ab3526, Abcam)]. Samples were washed with 0.02 % Tween-20 (#P7949, Sigma-Aldrich) in PBS (TPBS) and incubated with the secondary antibody\u0026nbsp;[Goat anti Mouse Alexa Flour 568 (#A11004, ThermoFisher) and Goat anti Rabbit Alexa Flour 488 (#A11008, ThermoFisher)] or BODIPY\u003csup\u003e™\u003c/sup\u003e 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) 1/1000 to stain the neutral lipids in PBS for 30 minutes at 37°C. The samples were washed three times with TPBS and mounted using ProLong™ Gold Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI) (#P36931, ThermoFisher).\u003c/p\u003e\n\u003cp\u003eThe images were captured using Zeiss AXIO Observer Z1 inverted microscope and Zeiss Zen pro software (Carl Zeiss). Videos were recorded with a Nikon Confocal A1R+ (Nikon) microscopy and Nikon Imaging Software (NIS) Elements Advanced Research Software version 5.30.02 (Nikon, Tokyo, Japan). The fluorescence signal of the immunoassayed proteins (α-SMA, Laminin, MyH1) in the spheroids was calculated as the percentage of area occupied by the signal of the target protein per total area of the image (in pixels) and normalized by the area of the spheroid (DAPI signal) using a macro for Image J software \u003csup\u003e31\u003c/sup\u003e developed and kindly provided by Benedicte Chazaud, Institute NeuroMyoGène, Lyon, France.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSuborbital flight on board of a microgravitational vehicle.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D models of MSCs and IMAT were transferred into 2ml cryotubes containing their respective media. A custom-build cartridge (Swedish Space Corporation, Sweden) was used to secure the 3D models into the Mapheous 14 sounding rocket shared module, launched from Esrange Space Center (Kiruna, Sweden) on the 27th of February of 2024. Six IMAT and six MSC 3D models were subjected to spaceflight. The same number of 3D models remained in the lab of Esrange Space Center as ground controls. Following the suborbital flight, the samples were recovered (within 2 hours after landing), all 3D models were promptly washed in PBS, resuspended in TRIzol to preserve the RNA, froze in dry ice, and shipped in dry ice to the laboratories of the Space \u0026amp; Environmental Physiology group at Karolinska Institutet for transcriptional analysis (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRNA extraction and sequencing and transcriptional analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D models’ total RNA was extracted following the column-based protocol previously described \u003csup\u003e29\u003c/sup\u003e.\u0026nbsp;SMART-Seq libraries were synthesized from total RNA using SMART-Seq® v4 Ultra® Low Input RNA Kit (TaKaRa Bio USA, Mountain View, CA, USA) according to the manufacturer’s instructions. The first-strand cDNAs were synthesized from the total RNA using nontemplated nucleotides added by the SMARTScribe™ Reverse Transcriptase, then, the double-strand cDNAs were synthesized by template switching.\u0026nbsp;PCR was directly performed and samples purified. Quality control of cDNAs were performed on Agilent Tapestation according to the manufacturer’s instructions. 1 ng of cDNA was used for the tagmentation reaction. During tagmentation, samples are fragmented and tagged with adapters, and a PCR is done to amplify the tagged DNA and sequencing indexes are added. The indexed libraries were purified, normalised and combined. The pool was sequenced on the Illumina Nextseq 2000 on a P2 100 flowcell sequencing run, generating 2x60 bp paired end reads. Bcl files were converted and demultiplexed to fastq using the bcl2fastq program. STAR v2.7.11b was used to index the human reference genome (hg38/GRCh38) and align the resulting fastq files. Mapped reads were then counted in annotated exons using featureCounts. The gene annotations (Homo_sapiens.GRCh38.113.gtf) and reference genome were obtained from Ensembl. The count table from featureCounts was imported into R/Bioconductor and differential gene expression was performed using the EdgeRpackage and its general linear model’s pipeline. For the gene expression analysis genes were filtered using the filterByExpr function and normalized using TMM normalization. Genes with an FDR adjusted p value \u0026lt;0.05 were termed significantly regulated. GO and pathway analysis was conducted using the goana, kegga and camera\u0026nbsp;functions from the limma packagem, while Wikipathways were retrieved using the rWikiPathways R library.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using unpaired t-tests between the 3D models exposed to for ten days to adipogenic medium (+AM) and control (-AM; kept in maintenance medium). Outliers were detected and removed using the Tukey test. Figure 1 statistical procedures and graphs were carried out in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A significance threshold of 5% (p-value \u0026lt; 0.05) was used.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP-D: experimental design, laboratory work and methods, data analysis and visualization, interpretation of the results, funding, manuscript drafting and review. JGL: laboratory work and methods, manuscript review. HR: laboratory work and methods, data visualization, manuscript review. AD: transcriptional methods and analysis, manuscript review. TG: interpretation of the results, manuscript review. RF-G: experimental design, interpretation of the results, funding, manuscript drafting and review, project coordination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments included in this study were performed according to principles of the declaration of Helsinki and approved by the Swedish Ethical Review Authority (#2022-04662-01, with addendums #2023-05072-02 and #2023-05072-02). Samples were obtained with written informed consent. The study is registered in ClinicalTrials.gov, with registration number\u0026nbsp;NCT05801185.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has been funded by Gösta Fraenckels stiftelse, with grants to SPD (#2023-01797 and #2024-02295) and RFG (#2023-01774 and #2024-02297). RFG is supported by a career grant from the Swedish National Space Agency (#2021-00159). SPD is supported by IBSA foundation fellowship 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets and material analyzed in this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to for the invaluable technical assistance made by BEA (Bioinformatics and Expression Analysis) core facility at Novum, Karolinska Institutet in Huddinge (Sweden). Likewise, the authors thank the Swedish Space corporation (SSC) for their helpful operational contribution to the study, in particular Stefan Krämer, Gunnar Florin, and Marton Galbacs.\u0026nbsp;The authors declare that they have not used Artificial Intelligence in this study.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWilliams, D., Kuipers, A., Mukai, C., and Thirsk, R. (2009). Acclimation during space flight: effects on human physiology. CMAJ \u003cem\u003e180\u003c/em\u003e, 1317\u0026ndash;1323. https://doi.org/10.1503/CMAJ.090628.\u003c/li\u003e\n \u003cli\u003eLee, S.J., Huynh, T. V., Lee, Y.S., Sebald, S.M., Wilcox-Adelman, S.A., Iwamori, N., Lepper, C., Matzuk, M.M., and Fan, C.M. (2012). Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway. 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A simplified and defined serum-free medium for cultivating fat across species. iScience \u003cem\u003e26\u003c/em\u003e, 105822. https://doi.org/10.1016/J.ISCI.2022.105822.\u003c/li\u003e\n \u003cli\u003eAgley, C.C., Velloso, C.P., Lazarus, N.R., and Harridge, S.D.R. (2012). An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents: Application to the Measurement of Human Muscle Cells in Culture. Journal of Histochemistry and Cytochemistry \u003cem\u003e60\u003c/em\u003e, 428. https://doi.org/10.1369/0022155412442897.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Skeletal muscle, Mesenchymal stem cells, Intramuscular adipose tissue, Suborbital flight, Space stressors","lastPublishedDoi":"10.21203/rs.3.rs-7745208/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7745208/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Spaceflight negatively impacts skeletal muscle mass and function, and current countermeasures fail to completely offset those alterations. Innovative approaches are then needed to understand the mechanisms governing space-induced muscle changes. This study explores short-term microgravity (suborbital flight) effects on transcriptional profile of skeletal muscle mesenchymal cells and intramuscular adipose tissue. We identified a set of molecular factors and biological processes impacted by microgravity in these cell types, warranting further investigation.","manuscriptTitle":"Transcriptomic Profiling of Muscle-Derived Stromal and Adipocytes Cells Exposed to Suborbital Microgravity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 08:47:11","doi":"10.21203/rs.3.rs-7745208/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-20T15:48:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213713495307657736449599466887163953736","date":"2026-05-06T16:39:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-04T15:08:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T14:32:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-07T06:35:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Microgravity","date":"2025-09-29T20:34:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7c880f3-86ec-4e60-be20-d0830d651890","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-20T15:48:43+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"213713495307657736449599466887163953736","date":"2026-05-06T16:39:14+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56285081,"name":"Biological sciences/Cell biology"},{"id":56285082,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-04T15:23:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 08:47:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7745208","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7745208","identity":"rs-7745208","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}