Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells

Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/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 Research Article Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells Damien Veret, Gautier Tejedor, Esther PEREZ, Alison Chomette, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4690241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jan, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 4 You are reading this latest preprint version Abstract Background: The regenerative potential of mesenchymal stromal/stem cells (MSCs) has been extensively studied in clinical trials over the past decade. Despite the promising regenerative properties documented in preclinical studies, such as those for osteoarthritis (OA), the therapeutic translation of these results in patients has not been fully conclusive. One factor contributing to this therapeutic barrier could be the presence of senescent cells in the OA joint. Methods: This study explores a novel approach to the treatment of OA by combining the therapeutic potential of adipose tissue-derived MSCs (AD-MSCs) with Rapamycin, a clinically approved immunosuppressive drug with anti-senescent properties. We first investigated the effects of Rapamycin on senescence and fibrosis markers in freshly isolated OA chondrocytes by immunostaining. We next evaluated the in vitro differentiation capacities of AD-MSCs, their regulatory immune functions on activated immune cells and their regenerative effects on OA chondrocyte signature in presence of Rapamycin. Results: Rapamycin was found to reduce in OA chondrocytes the senescence marker p15ink4b and the fibrotic marker COL1A1 in a dose-dependent manner without affecting the expression of the master chondrogenic markers SOX9 and COL2. The drug enhanced the differentiation of AD-MSCs into chondrocytes and reduced their adipogenesis. In addition, Rapamycin improved the immunoregulatory functions of AD-MSCs by promoting the expression of immunosuppressive elements such as IDO1, PTGS2, and PDL-1/CD274, the latter at the cell surface. Finally, by comparative RNA sequencing analysis, we revealed that AD-MSCs in presence of Rapamycin exhibited an improved chondroprotective regenerative effects on cocultivated OA chondrocytes. Conclusions: Our findings propose that the combination of Rapamycin and AD-MSCs enhances the therapeutic efficacy of these cells on senescence-driven degenerative diseases such as OA. Overall, our data also open avenue for the identification of some factors that could contribute to the enhanced anti-fibrotic and anti-inflammatory properties of AD-MSCs in response to Rapamycin. mesenchymal stromal/stem cells Rapamycin regenerative medicine osteoarthritis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Supplementary Files SuppFigure1.pdf Supplementary Figure 1: Rapamycin has no effect on AD-MSC self-renewal. Primary AD-MSCs from healthy donors at passage 1 were seeded at 100 cells/9.6cm 2 (~10 cells/cm 2 ) in the presence (or absence) of different concentrations of Rapamycin. Media were not removed along the experiment. After 10 days culture, colony were fixed, stained with Giemsa and counted. 0.01% DMSO (vehicle) treatment was used as a control. Mean effect on n=3 different AD-MSC donors, each performed in triplicate. Data were plotted and analyzed using GraphPad Prism 10.2.3 by Two-way ANOVA. (p-value: * <0.05, ** < 0.01, ***<0.001). SuppFigure2.pdf Supplementary Figure 2: Rapamycin does not significantly affect the expression of adipogenic markers. AD-MSCs in adipogenic and control conditions were treated the first 2 days after the induction of differentiation with 10nM rapamycin or 0.01% DMSO as vehicle control. At day 10, cells were lysed and the expression of PPARG2 , FABP4 , LEPTIN and ADIPOG were analysed by RTqPCR. RPLP0 was used as housekeeping genes. All error bars represent SEM and statistical analysis was performed by 2-way ANOVA using GraphPad Prism 102.3 (p-value: *<0.05, **<0.01, ***<0.001). SuppFigure3.pdf Supplementary Figure 3: RNAseq analysis reveals that Rapamycin sensitizes AD-MSCs to IFN-g response. (A) REACTOME pathway of all genes significantly induced by IFNγ treatment (FDR ≤ 0.05). (B) REACTOME pathway of the n=81 genes commonly induced by IFN-γ alone and IFN-γ+RAPA treatments (from the Venn diagram in Figure 3B), FDR ≤ 0.05. SuppFigure4.pdf Supplementary Figure 4: Defining OA versus healthy cartilage signature from GSE114007. Raw counts table from GSE114007 were retrieved and reanalysed using EdgeR . Reanalysis led to a list of 1956 upregulated and 1878 downregulated genes at FDR ≤ 0.05. (A) The n=1956 upregulated genes in OA were submitted to KEGG disease databank and significantly affected pathways (q-value ≤ 0.05) were presented as a bubble chart. The entire list of dysregulated genes in OA was submitted to KEGG pathway (B) and REACTOME (C), presented as a dual-sided histograms showing the number of genes affected in each significantly affected pathway (FDR ≤ 0.05). (D) Dot plots showing the normalized expression (log10(normalized counts+1)) in the n=18 healthy and n=20 OA cartilages of chondrogenic ( SOX9 ), osteogenic ( ALPL, SPARC, POSTN) and fibrotic ( COL1A1, COL3A1, COL5A1, ELN) markers. Statistical comparisons were performed using Mann-Withney test. (P-value: * <0.05, **<0.01, ***<0.001). Cite Share Download PDF Status: Published Journal Publication published 15 Jan, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 17 Jul, 2024 Reviewers invited by journal 17 Jul, 2024 Editor assigned by journal 05 Jul, 2024 First submitted to journal 05 Jul, 2024 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-4690241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328163772,"identity":"ee318863-e4da-45f1-b55a-71a6e0b066e0","order_by":0,"name":"Damien Veret","email":"","orcid":"","institution":"INSERM U1183","correspondingAuthor":false,"prefix":"","firstName":"Damien","middleName":"","lastName":"Veret","suffix":""},{"id":328163773,"identity":"507ce14c-d6e6-4df5-9124-c69ed0e03668","order_by":1,"name":"Gautier Tejedor","email":"","orcid":"","institution":"Montpellier Life 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Hospital Center of Montpellier: Centre Hospitalier Universitaire de Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Yves-Marie","middleName":"","lastName":"Pers","suffix":""},{"id":328163780,"identity":"847c11e3-f0c5-4a19-96ea-56965897b4b5","order_by":8,"name":"Christian Jorgensen","email":"","orcid":"","institution":"Regional University Hospital Center of Montpellier: Centre Hospitalier Universitaire de Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Jorgensen","suffix":""},{"id":328163781,"identity":"19bb27f5-9dd2-440e-9a58-96efc492c678","order_by":9,"name":"Claire Gondeau","email":"","orcid":"","institution":"Montpellier Life Science","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Gondeau","suffix":""},{"id":328163782,"identity":"1081f8cf-7638-453b-8daa-0d7df23dfcb4","order_by":10,"name":"jean-marc Brondello","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5991-3945","institution":"Institut national de la santé et de la recherche médicale: INSERM","correspondingAuthor":true,"prefix":"","firstName":"jean-marc","middleName":"","lastName":"Brondello","suffix":""}],"badges":[],"createdAt":"2024-07-05 07:00:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4690241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4690241/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-024-04090-8","type":"published","date":"2025-01-15T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62656575,"identity":"276ccc5c-2175-491e-855c-4ba0422d370d","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2709749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapamycin exerts anti-fibrotic and anti-senescent effects on primary OA chondrocytes. \u003c/strong\u003ePrimary chondrocytes were isolated from OA cartilages (n=6), cultured at 1.5 x 10\u003csup\u003e5\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e (nearly confluent state) for 7 to 10 days in FBS-containing medium, passaged at 1.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e, and treated 24h later with 10, 50 or 100 nM Rapamycin for 48h. Exposure to 0.01% DMSO (vehicle) was used as control. \u003cstrong\u003e(A) \u003c/strong\u003eConfocal microscopy and \u003cstrong\u003e(B) \u003c/strong\u003equantification of SOX9 and p15 co-staining in OA chondrocytes. \u003cstrong\u003e(C) \u003c/strong\u003eConfocal microscopy and \u003cstrong\u003e(D) \u003c/strong\u003equantification of COL2 and COL1A1 co-staining in OA chondrocytes. Data were plotted and analysed using GraphPad Prism 10.2.3 by Two-way ANOVA. (p-value: * \u0026lt;0.05, ** \u0026lt; 0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/112de1e28ccc6d6facd40c9c.png"},{"id":62656577,"identity":"6bd820d2-fb8d-4b10-baf4-f95e7cccb618","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2536138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Rapamycin on AD-MSC differentiation characteristics. \u003c/strong\u003eAD-MSCs at passage 1 were seeded at 4.5x10\u003csup\u003e3\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e for 4 days before induction of differentiation using STEMCELL MesencultTM kits. Cells were exposed to 10nM rapamycin or 0.01% DMSO during the first two days of differentiations. Adipogenesis (n=4) was stopped after 10 days, osteogenesis (n=4) after 14 days and chondrogenesis (n=3) after 21 days. (\u003cstrong\u003eA\u003c/strong\u003e) Adipogenesis visualization by Oil Red O staining. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of Oil Red O staining by measuring the absorbance at 540 nm. \u003cstrong\u003e(C) \u003c/strong\u003eConfocal microscopy showing the expression of the adipogenic transcription factor PPARγ in undifferentiated and differentiated AD-MSCs and \u003cstrong\u003e(D) \u003c/strong\u003equantification of the number of PPARγ positive cells in differentiated AD-MSCs (left). The nuclear mRNA expression level of PPARγ was evaluated by RTqPCR (right). \u003cstrong\u003e(E) \u003c/strong\u003eOsteogenis visualization by Alizarin red staining and \u003cstrong\u003e(F) \u003c/strong\u003equantification by measuring the absorbance at 540 nm. \u003cstrong\u003e(G) \u003c/strong\u003eChondrogenic pellets stained with alcian blue. \u003cstrong\u003e(H) \u003c/strong\u003eRelative quantification of \u003cem\u003eSOX9 \u003c/em\u003eand \u003cem\u003eACAN \u003c/em\u003ecartilage markers by RTqPCR in chondrogenic pellets. All error bars represent SEM and statistical analysis were performed by 2-way ANOVA using GraphPad Prism 10.2.3 (p-value: *\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/1308a1c1a20d1ff82950a334.png"},{"id":62657527,"identity":"9881377a-918c-46a6-ab62-62ed364a9f93","added_by":"auto","created_at":"2024-08-17 02:10:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":510194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapamycin and AD-MSCs exert additive immunosuppressive effects. \u003c/strong\u003eThe expression profiles of AD-MSCs (n=4 different donors) treated with 10nM Rapamycin or 0.01% DMSO, upon IFN-γ stimulation or resting conditions were analysed by RNAseq. Cells were seeded at 1.5x10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e for 24h and then treated for 48h. \u003cstrong\u003e(A) \u003c/strong\u003eVolcano Plot showing all genes induced or repressed upon IFN-γ treatment. \u003cstrong\u003e(B) \u003c/strong\u003e2-way Venn diagram representing genes commonly induced by IFNγ treatment (\u003cem\u003evs \u003c/em\u003eCTRL_Veh.) (green circle) and by IFNγ+RAPA (\u003cem\u003evs \u003c/em\u003eIFNg) (orange circle). \u003cstrong\u003e(C) \u003c/strong\u003eHeatmap representing the n=81 genes commonly overexpressed by IFNg and IFNg+RAPA treatments. \u003cstrong\u003e(D, E, F) \u003c/strong\u003eTranscripts Per Million (TPM) of \u003cem\u003eIDO1\u003c/em\u003e, \u003cem\u003ePTGS2\u003c/em\u003e, and \u003cem\u003eCD274 \u003c/em\u003e(PD-L1), respectively. \u003cstrong\u003e(G) \u003c/strong\u003ePD-L1 protein overexpression determined by flow cytometry. \u003cstrong\u003e(H) \u003c/strong\u003eAssessment by flow cytometry of the proliferation of PHA-stimulated PBMC (\u003cem\u003evs \u003c/em\u003eUnstimulated) alone or with 2 different ratios of AD-MSCs (1:10 and 1:20), in presence or not of 10nM rapamycin. PBMC were stained with CellTrace Violet and stimulated with PHA (5 ng/mL) for 4 days. Overlayed histograms (left) and quantification of the PBMC proliferation. (right). All error bars represent SEM and statistical analysis were performed by 2-way ANOVA using GraphPad Prism 10.2.3 (p-value: *\u0026lt;0,05, **\u0026lt;0,01, ***\u0026lt;0,001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/b23a36c44e0e5bd42a147575.png"},{"id":62656578,"identity":"42addfed-5a6c-4f82-ba62-8acbe03dba64","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":511086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImproved therapeutic effects of AD-MSCs in presence of Rapamycin on OA chondrocytes phenotype\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003eSchematic workflow showing the cultures of OA chondrocytes (n=3 donors) alone or with AD-MSCs (n=3 donors) in Trans-well, in the presence of 10nM rapamycin or 0.01% DMSO as vehicle for 72h. The comparison groups are the following: CH+RAPA/CH+Veh. (referred as “RAPA”), CH+AD-MSC/CH+Veh. (“ADSC”) and CH+RAPA+AD-MSC/CH+AD-MSC (referred as “COMBI”). \u003cstrong\u003e(B) \u003c/strong\u003eHistograms showing the number of differentially expressed genes (DEG) in each comparison group. \u003cstrong\u003e(C) \u003c/strong\u003e4-way Venn diagram and associated chart representing the number of genes modulated by the COMBI compared to GSE114007 reanalysis (in %) from supplementary figure 4. \u003cstrong\u003e(D) \u003c/strong\u003eDual-sided histogram showing the \u003cem\u003eKEGG pathways \u003c/em\u003esignificantly dysregulated (FDR ≤ 0.05) in OA (from supplementary figure 4, plain bars) merged with the corresponding genes of each pathway affected by COMBI. (dashed bars). \u003cstrong\u003e(E) \u003c/strong\u003eDual-sided histogram showing the \u003cem\u003eREACTOME \u003c/em\u003epathways significantly dysregulated ((FDR ≤ 0.05) in OA (discovered from GSE114007 dataset reanalysis) (plain bars) merged with the corresponding genes of each pathway affected by COMBI. (dashed bars). (\u003cstrong\u003eF\u003c/strong\u003e) Line plot representing Transcripts Per Million (TPM) for chondrogenic (\u003cem\u003eSOX9\u003c/em\u003e), hypertrophic (\u003cem\u003eALPL, SPARC, POSTN\u003c/em\u003e) and fibrotic markers (\u003cem\u003eCOL1A1, COL3A1, COL5A1)\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/987cc3642c7bd12d04213fcf.png"},{"id":62656580,"identity":"f43a606f-db41-4cd0-bdc6-7302cd765d45","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1049677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapamycin induces the expression of chondroprotective and regenerative factors by AD-MSCs. (A) \u003c/strong\u003eThe list of rapamycin-upregulated genes (\u003cem\u003evs \u003c/em\u003evehicle) in AD-MSCs was submitted to \u003cem\u003eKEGG pathway\u003c/em\u003e. Significantly affected pathways (at FDR ≤ 0.05) were plotted as a bubble chart. \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eKEGG network \u003c/em\u003eof \u0026lt;\u0026lt; Hedgehog signaling \u0026gt;\u0026gt; and \u0026lt;\u0026lt; TGF-β signaling \u0026gt;\u0026gt;upregulated genes identified in panel A. \u003cstrong\u003e(C) \u003c/strong\u003eBoth upregulated and downregulated genes encoding extracellular factors in AD-MSCs were selected by using the \u003cem\u003eGene Ontology « Extracellular region » \u003c/em\u003ereference (GO0005576) and plotted as a Z-score Heatmap of normalized counts showing vehicle- \u003cem\u003evs. \u003c/em\u003erapamycin-treated groups. \u003cstrong\u003e(D) \u003c/strong\u003eTranscripts Per Million (TPM) of extracellular factors from the \u0026lt;\u0026lt; Hedgehog signaling \u0026gt;\u0026gt; \u003cem\u003e(SCUBE2 \u003c/em\u003eand \u003cem\u003eMEGF8\u003c/em\u003e) and the \u0026lt;\u0026lt; TGF-β signaling \u0026gt;\u0026gt; (\u003cem\u003eLTBP2 \u003c/em\u003eand \u003cem\u003eFST\u003c/em\u003e) were extracted and plotted as dot-histograms to show the relative expression among groups. \u003cstrong\u003e(E) \u003c/strong\u003eTPM of the pro-regenerative factor GAS6 extracted and plotted. (\u003cstrong\u003eF\u003c/strong\u003e) Relative secretion of GAS6 by AD-MSCs upon exposure to Rapamycin, IFN-γ, alone or in co-treatment. GAS6 protein level in supernatant was quantified by ELISA and normalized by the relative number of cells. Data were plotted and analysed using GraphPad Prism 10.1 by Two-way ANOVA. (p-value: * \u0026lt;0.05, ** \u0026lt; 0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/5d89b871e07ac6dff9059c66.png"},{"id":74284480,"identity":"cbc883d0-a029-49e7-a747-33989cfe1e6c","added_by":"auto","created_at":"2025-01-20 16:07:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2940777,"visible":true,"origin":"","legend":"","description":"","filename":"VeretdetalsubmittedFINAL2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1_covered_fbfab843-c60a-4583-8012-5b24fa54e036.pdf"},{"id":62656574,"identity":"d647c679-9f5f-431d-be51-0eacc7d6178d","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1: Rapamycin has no effect on AD-MSC self-renewal. \u003c/strong\u003ePrimary AD-MSCs from healthy donors at passage 1 were seeded at 100 cells/9.6cm\u003csup\u003e2\u003c/sup\u003e (~10 cells/cm\u003csup\u003e2\u003c/sup\u003e) in the presence (or absence) of different concentrations of Rapamycin. Media were not removed along the experiment. After 10 days culture, colony were fixed, stained with Giemsa and counted. 0.01% DMSO (vehicle) treatment was used as a control. Mean effect on n=3 different AD-MSC donors, each performed in triplicate. Data were plotted and analyzed using GraphPad Prism 10.2.3 by Two-way ANOVA. (p-value: * \u0026lt;0.05, ** \u0026lt; 0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"SuppFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/0f05b652b464a0f6b62a72d7.pdf"},{"id":62656576,"identity":"c712283f-bd5b-4d34-8a29-cfa97a356766","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":61171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2: Rapamycin does not significantly affect the expression of adipogenic markers. \u003c/strong\u003eAD-MSCs in adipogenic and control conditions were treated the first 2 days after the induction of differentiation with 10nM rapamycin or 0.01% DMSO as vehicle control. At day 10, cells were lysed and the expression of \u003cem\u003ePPARG2\u003c/em\u003e, \u003cem\u003eFABP4\u003c/em\u003e, \u003cem\u003eLEPTIN \u003c/em\u003eand \u003cem\u003eADIPOG \u003c/em\u003ewere analysed by RTqPCR. \u003cem\u003eRPLP0 \u003c/em\u003ewas used as housekeeping genes. All error bars represent SEM and statistical analysis was performed by 2-way ANOVA using GraphPad Prism 102.3 (p-value: *\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"SuppFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/ac5f3a8e3ac986ad0880920c.pdf"},{"id":62656582,"identity":"955ad5e2-3d0a-4463-b431-ed8a7e05b81a","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":88763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3: RNAseq analysis reveals that Rapamycin sensitizes AD-MSCs to IFN-\u003c/strong\u003eg \u003cstrong\u003eresponse\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eREACTOME \u003c/em\u003epathway of all genes significantly induced by IFNγ treatment (FDR ≤ 0.05). \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eREACTOME \u003c/em\u003epathway of the n=81 genes commonly induced by IFN-γ alone and IFN-γ+RAPA treatments (from the Venn diagram in Figure 3B), FDR ≤ 0.05.\u003c/p\u003e","description":"","filename":"SuppFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/f2f54971ff034381e79cfc5c.pdf"},{"id":62656579,"identity":"c962365b-8cf7-4923-b1a2-71a890fe127c","added_by":"auto","created_at":"2024-08-17 02:02:41","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":324609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4: Defining OA versus healthy cartilage signature from GSE114007. \u003c/strong\u003eRaw counts table from GSE114007 were retrieved and reanalysed using \u003cem\u003eEdgeR\u003c/em\u003e. Reanalysis led to a list of 1956 upregulated and 1878 downregulated genes at FDR ≤ 0.05. \u003cstrong\u003e(A) \u003c/strong\u003eThe n=1956 upregulated genes in OA were submitted to \u003cem\u003eKEGG \u003c/em\u003edisease databank and significantly affected pathways (q-value ≤ 0.05) were presented as a bubble chart. The entire list of dysregulated genes in OA was submitted to \u003cem\u003eKEGG pathway \u003c/em\u003e\u003cstrong\u003e(B) \u003c/strong\u003eand \u003cem\u003eREACTOME \u003c/em\u003e\u003cstrong\u003e(C)\u003c/strong\u003e, presented as a dual-sided histograms showing the number of genes affected in each significantly affected pathway (FDR ≤ 0.05). \u003cstrong\u003e(D) \u003c/strong\u003eDot plots showing the normalized expression (log10(normalized counts+1)) in the n=18 healthy and n=20 OA cartilages of chondrogenic (\u003cem\u003eSOX9\u003c/em\u003e), osteogenic (\u003cem\u003eALPL, SPARC, POSTN) \u003c/em\u003eand fibrotic (\u003cem\u003eCOL1A1, COL3A1, COL5A1, ELN) \u003c/em\u003emarkers. Statistical comparisons were performed using Mann-Withney test. (P-value: * \u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"SuppFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690241/v1/0383b9a29e028515346159ce.pdf"}],"financialInterests":"","formattedTitle":"Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mesenchymal stromal/stem cells, Rapamycin, regenerative medicine, osteoarthritis","lastPublishedDoi":"10.21203/rs.3.rs-4690241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4690241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The regenerative potential of mesenchymal stromal/stem cells (MSCs) has been extensively studied in clinical trials over the past decade. Despite the promising regenerative properties documented in preclinical studies, such as those for osteoarthritis (OA), the therapeutic translation of these results in patients has not been fully conclusive. One factor contributing to this therapeutic barrier could be the presence of senescent cells in the OA joint.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study explores a novel approach to the treatment of OA by combining the therapeutic potential of adipose tissue-derived MSCs (AD-MSCs) with Rapamycin, a clinically approved immunosuppressive drug with anti-senescent properties. We first investigated the effects of Rapamycin on senescence and fibrosis markers in freshly isolated OA chondrocytes by immunostaining. We next evaluated the in vitro differentiation capacities of AD-MSCs, their regulatory immune functions on activated immune cells and their regenerative effects on OA chondrocyte signature in presence of Rapamycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Rapamycin was found to reduce in OA chondrocytes the senescence marker p15ink4b\u003c/p\u003e\n\u003cp\u003eand the fibrotic marker COL1A1 in a dose-dependent manner without affecting the expression of the master chondrogenic markers SOX9 and COL2. The drug enhanced the differentiation of AD-MSCs into chondrocytes and reduced their adipogenesis. In addition, Rapamycin improved the immunoregulatory functions of AD-MSCs by promoting the expression of immunosuppressive elements such as IDO1, PTGS2, and PDL-1/CD274, the latter at the cell surface. Finally, by comparative RNA sequencing analysis, we revealed that AD-MSCs in presence of Rapamycin exhibited an improved chondroprotective regenerative effects on cocultivated OA chondrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur findings propose that the combination of Rapamycin and AD-MSCs enhances the therapeutic efficacy of these cells on senescence-driven degenerative diseases such as OA. Overall, our data also open avenue for the identification of some factors that could contribute to the enhanced anti-fibrotic and anti-inflammatory properties of AD-MSCs in response to Rapamycin.\u003c/p\u003e","manuscriptTitle":"Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-17 02:02:36","doi":"10.21203/rs.3.rs-4690241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-17T10:16:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T09:43:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T13:30:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2024-07-05T08:48:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87c3fa24-7f9d-404c-9ef2-ead725672eb4","owner":[],"postedDate":"August 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T15:59:17+00:00","versionOfRecord":{"articleIdentity":"rs-4690241","link":"https://doi.org/10.1186/s13287-024-04090-8","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-01-15 15:57:01","publishedOnDateReadable":"January 15th, 2025"},"versionCreatedAt":"2024-08-17 02:02:36","video":"","vorDoi":"10.1186/s13287-024-04090-8","vorDoiUrl":"https://doi.org/10.1186/s13287-024-04090-8","workflowStages":[]},"version":"v1","identity":"rs-4690241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4690241","identity":"rs-4690241","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}