The effect of 2D culture on stemness-related transcriptome in shed microvesicles of mesenchymal stem cells

The effect of 2D culture on stemness-related transcriptome in shed microvesicles of mesenchymal stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effect of 2D culture on stemness-related transcriptome in shed microvesicles of mesenchymal stem cells Fatemeh Foroughi Fard, Behnaz Bakhshandeh, Zohreh Jahanafrooz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2842807/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Over the past years, the applications of mesenchymal stem cells (MSCs) grow rapidly in medicine as a promising option for regenerative medicine. So, increasing demands for MSCs and their derivations as cell-based drugs in the market are anticipated. One of the major challenges in MSC therapy is possible alterations in the stemness potential of MSCs during in vitro culture and proliferation. Microvesicles (MVs) reflect the transcriptional status of cells of origin. This study aimed to investigate the impact of 2D culture on stemness characteristics of MSCs by focusing on transcriptome in shed MVs. Methods and Results MVs were isolated from rat bone marrow MSCs using ultracentrifugation. Transmission electron microscopy and dynamic light scattering were applied to characterizing MVs. Then, some markers related to stemness and differentiation were evaluated in shed MVs during 10-day culture. Meanwhile the cell proliferation and apoptosis were assessed in cultured MSCs. Based on our results, not only MSCs-derived MVs preserved somewhat their stemness contents but also MSCs maintained proliferative during 10-day 2D culture. Conclusion Taken together, 2D culture had no significant effect on the biological state of MSCs which strengthened their applications in cell therapies. Further studies are needed to identify the protein component of MVs to give some benefits for biomimetic approaches. Microvesicles Mesenchymal stem cells Apoptosis Stemness potential 2D culture Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Stem cells have self-renewal and differentiation capacities, which make them promising candidates for cell therapy. Embryonic stem cells (ESCs), MSCs, hematopoietic stem cells, and induced pluripotent stem cells (iPSCs) are major categories of stem cells [ 1 ]. MSCs are found in the most fetus and adult tissues, which are easily available compared to other stem cell types. MSCs have immunomodulatory properties with potential differentiation capacity into diverse cell types (i.e., both mesenchymal and ecto/endodermal lineages). The ability to migrate into injured tissues and direct initiation of tissue repair makes MSCs more favorable in regenerative medicine [ 2 , 3 ]. MSCs have a tropism to tumor tissue and can be used as a cell-based anticancer drug [ 4 ]. Releasing of vesicles, including MVs, exosomes, and apoptotic bodies is a conserved process and a common way of cell-to-cell communication and signaling with tissue-specific contents in almost all cell types [ 5 – 7 ]. Exosomes are released by cells upon fusion of multi-vesicular bodies with the plasma membrane while MVs are directly bud from the plasma membrane and transport protein, lipids, mRNA, miRNA, and membrane receptors among the cells [ 8 , 9 ]. Recently, many researches have been focused on the properties of MVs such as small size (i.e., less than 1µm), high flexibility, capability to cross the biological barrier, and protecting capacity of their cargo, to acquire them in therapeutic application such as drug delivery, favorable differentiation and reshaping the injured tissues. MVs derived from stem cells are promising mediators in regenerative medicine. Stem cell-derived MVs therapy is a type of stem cell-based therapies, which provides a safer treatment than whole stem cells [ 10 , 11 ]. Mohammadi et al. confirmed the immunomodulatory effects of human MSCs-derived MVs, and the role of MVs as an alternative for MSCs therapy [ 8 ]. Recent successful translations of stem cell-based therapies to patients have enriched the idea that such strategies may one day become a treatment in regenerative medicine [ 12 ]. However, stemness maintenance during in vitro culture is a key issue for stem cell-based applications in cell therapies. Actually, in vitro condition and especially 2D culture cannot mimic the natural 3D situation of the body including biochemicals (such as growth factors, cytokines, extracellular components, and microbial-derived compounds) and biophysicals (such as surface topographic textures and mechanical cues) [ 13 ]. Moreover, repetitive in vitro cell passages accelerate MSCs senescence leading to decreased stemness state [ 14 ]. Therefore, many studies have evaluated the ways to preserve the stemness capacity of stem cells during in vitro cultures [ 15 ]. Different strategies such as organoid culture, genetic manipulation, optimized cell culture methods, and utilization of biochemical and biophysical signals have been applied for this purpose [ 16 , 17 ]. In spite of undesirable effects of 2D culture, due to cost beneficially and simplicity, 2D culture is a well-established and widely used method compared to 3D culture. On the other hand, the developing applications of MVs have prompted us to evaluate the possible changes of MSCs-derived MVs content during 2D culture. Gene expression is a valuable molecular marker associated with stemness characteristic of MSCs. Overexpression of genes involved in embryonic-like phenotype along with downregulation of differentiation related genes can confer the maintenance of stemness state of MSCs [ 15 , 18 ]. Therefore, herein the transcriptional alterations of some stem cell-associated markers namely OCT4 and ALP as well as some differentiation related genes such as SOX9, RunX2, and Bglap genes [ 19 , 20 ] inside the secreted MVs were evaluated during 2D culture of MSCs. Overall, the impact of 2D culture on stemness characteristics of MSCs was investigated through focusing on the mRNA content of the secreted MVs. 2. Materials and methods 2.1. Cell isolation and culture We isolated MSCs from the bone marrow of 20-day-old albino female Norway rats, which were obtained from the animal house of University of Tehran. All experimental protocols were performed under ethics approval obtained by the Iran Animal Care Committee. Also, experiments were done in accordance with the approved protocols, institutional guidelines for the care and use of laboratory animals and ARRIVE recommendations. Bone marrow cells were flushed out of the cavity of femurs and tibias, then cultured in DMEM medium (Gibco™) supplemented with 20% FBS (Gibco™) and 1% penicillin/streptomycin (pen/strep) antibiotics (Gibco™) at 37°C in 5% CO 2 under 90–95% humidity. We removed non-adherent cells 2 and 5 days after primary culture and changed the medium every other day until cells reached 90% confluence. The adherent MSCs were detached by trypsin, reseeded as new cultures, and expanded for further studies. The isolated MSCs were characterized by flow cytometry analyses of CD44, CD29, CD45 and CD34 markers. 2.2. Differentiation protocols To evaluate the lineage commitment of isolated cells, osteogenic, chondrogenic, and adipogenic differentiation studies were carried out. For osteogenic differentiation, cells were incubated in osteogenic medium (DMEM, 7–10 mM dexamethasone, 10% FBS, 50 µg/ml ascorbic acid biphosphate, 10 mM ß-glycerol phosphate) for 14 days. To investigate calcium mineralization in osteogenic differentiation, cultured cells were treated with 1% Alizarin Red (Sigma-Aldrich) in pH 4.1 for 10 min at room temperature and visualized under a light microscope. For adipogenic induction, the growth medium was supplemented with 107 mM dexamethasone, 0.5 mM IBMX, 0.2 mM indomethacin, 66 nM Insulin for 21 days. Then the cells were stained by Oil Red (Sigma-Aldrich) to detect the accumulated oil droplets. Chondrogenic differentiation was induced by 50 µg/mL ascorbic acid bisphosphate, 250 nM dexamethasone, 200 ng/20 mL basic fibroblast growth factor (bFGF), 1% ITS (6.25 mg/mL insulin, 6.25 mg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid), and 10 ng/mL transforming growth factor-beta (Sigma-Aldrich). To identify the presence of hyaluronic acid in the chondrogenic differentiated cells Alcian Blue dye (Sigma-Aldrich) was used. 2.3. Cell death analysis by Annexin V and propidium iodide (PI) flow cytometry To investigate the trends of cell apoptosis and necrosis upon 2D culture, PI and Annexin V-FITC (EXBIO, Vestec, Czech Republic) were utilized for double-staining of MSCs, followed by flow cytometry analysis in day 1 and day 10 of culture. 2.4. Isolation of MVs Cell cultures were replaced with the serum-free medium about 8 hours before centrifugation to minimize the FBS albumin interference. The supernatants were collected and cell-free supernatants were obtained by 2,000-rpm centrifugation for 10 minutes at 4°C. Apoptotic bodies and cell debris were excluded by 10,000 g centrifugation for 20 minutes at 4°C. MVs pallets were collected after 60,000 g centrifugation at 4°C for one hour twice. The pellets were used freshly. To indirect estimation of the concentration of isolated MVs, from protein concentration, we applied the Bradford assay. In this assay, protein molecules bind to Coomassie dye under acidic conditions resulting in a color change from brown to blue. Absorbance is measured at 595 nm following a short room temperature incubation. 2.5. Characterization of MVs To determine the size and morphology of MVs, isolated MVs were stained with 2% uranyl acetate on formvar-carbon-coated grids. After drying, transmission images were provided by placing the grid in an electron microscope (Philips). DLS technique was used to determine the size range and homogeneity of the isolated MVs by re-suspending the MVs pellet in PBS and analyzing by Malvern instrument (Malvern). 2.6. Quantitative transcriptional evaluation Total RNA was extracted from MSC-derived MVs on days 1 and 10 of 2D culture using RNX Plus reagent (Cinnagen). Then, reverse transcription of mRNAs was performed using a cDNA synthesized kit according to the manufacturer's instructions (Cinnagen). The real-time PCR reactions were conducted utilizing the SYBR Green master mix (Takara) in Rotor-Gene 6000 Real-Time Thermal Cycler (Corbett Research, Australia). The primer sequences for β2m (housekeeping gene), Alkaline phosphatase (ALP), OCT4, SOX9, Osteocalcin (or Bglap), and RunX2 are provided in supplementary table 1 . Relative Expression Software Tool (REST 2009, Corbett Research, Australia) was used to measure relative transcriptions through the 2 (−∆∆Ct) approach. 2.7. Statistical analysis All tests were performed at least in triplicate. Data from two groups were compared by the student’s two-tailed t-test. The outcomes were presented as mean ± SD. A p-value of < 0.05 was considered significant. 3. Results 3.1. Characteristics of the cells As shown in Fig. 1 (A, B, C), red calcium deposition is an indication of osteogenic differentiation while secreted hyaluronic acid is an indication of cartilaginous differentiation. Obvious fat bright red droplets represent the differentiation of MSCs into adipocyte cells. Isolated morphology of MSCs were fibroblastic and spindle-shaped. Analyses of their surface markers by flow cytometry confirmed expression of CD44 and CD29 while these cells did not express CD45 and CD34 (Fig. 1 D). 3.2. Cell viability assessment during 2D culture Flow cytometry analyses of annexin V and PI were performed to evaluate the effects of 2D culture on MSCs viability and the type of cell death. There was no significant difference in live, early apoptotic, late apoptotic, and necrotic cell numbers between day 1 and day 10 of the culture (Fig. 2 ). 3.3. Characteristics of isolated MVs According to Bradford assay, the average protein contents of MVs on day 1 and day 10 were 9.5 5 µg/µL and 18.5 µg/µL, respectively. As shown in Fig. 3 , the average diameter of MVs was less than 1 µm with a round shape, confirming the efficacy of the protocol for the isolation and purification of MVs. DLS analysis showed that the shedding MVs were distributed in the range from 100 nm to 1000 nm in diameter. 3.4. Quantitative evaluation of the mRNA content of the MVs during 2D culture Gene expression analysis by quantitative PCR showed that mRNAs contents of MSCs-derived MVs on day 10 had no significant changes compared to day 1. As depicted in Fig. 4 , the patterns of transcriptional changes in the ALP, OCT4, SOX9, Osteocalcin, and RunX2 genes were different. While ALP and OCT4 mRNAs slightly decreased on day 10, SOX9, Bglap, and RunX2 mRNAs showed the opposite pattern inside the MVs. 4. Discussion and future remarks Over the past years, the efficacy of MSCs, as the main players in cell therapy, has been reported in various clinical trials [21]. The application of MSCs in medicine is a rapidly evolving approach and is a promising option for regenerative medicine [8, 22, 23]. So, increasing demand for MSCs and their derivations as cell-based drugs in the market is anticipated. However, some drawbacks, such as the pro-tumorigenic effect of implanted MSCs, short-time viability after injection, reaching low portions of MSCs to damaged tissue after systemic administration, the immunogenicity of the differentiated MSCs, induction of allo-antibodies in repeated administration of MSCs, and induction of antibodies against fetal bovine serum (FBS) used in the MSC culture medium have hindered this issue [24]. In this regard, the application of MSCs derivations such as shed MVs can be considered as a safer approach. Cha et al. showed that human MSCs secret remarkable MVs, and confirmed the therapeutic capacity of MSC-derived MVs [11]. One of the major challenges in MSC therapy is a possible alteration in stemness potential of MSCs during in vitro culture and proliferation [25]. During MSCs in vitro culture, autonomous osteoblastic self-differentiation was reported [26, 27]. Since MVs carry various cargos such as mRNA, proteins, miRNA, bioactive lipids, and signaling nucleotides, investigation of shed MVs could reflect the situation of the origin cells [28, 29]. Herein, the alterations in stemness potential of MSCs during a 10-day 2D culture was investigated both from cell proliferative status and their transcriptome in secreted MVs. Efficient expansion of the MSCs while avoiding any culture-related phenotypic changes and cell death, is an important challenge in MSCs therapy [30, 31]. In our study, apoptotic cell death of MSCs during a 10-day 2D culture was negligible (Figure 2). A neglectable decrease in the percentage of viable cells during cell culture has been reported in many studies in accordance with our study [31]. For indirect inspection of 10-day cultured MSCs, their shed MVs were isolated. Characterization of isolated MVs from MSCs, including shape, diameter, and size distribution (Figure 3) was in accordance with previous studies [32, 33]. There are few studies about the mRNA, miRNA, and protein contents of MVs. Xie, et al. reported regulation of hematopoiesis by miRNAs in MSCs-derived MVs applied in ex vivo expansion of cord blood mononuclear cells [33]. In another study, MSCs-derived MVs enhanced the survival of renal cells both in vitro and in vivo [32]. Herein some stemness markers such as OCT4 and ALP [34] were evaluated. In addition, some differentiation markers such as Bglap and RunX2 (osteogenic potential markers [35, 36]) and SOX9 (a marker of chondrogenic differentiation [37]) were also investigated [35-37]. Based on our findings, MSCs-derived MVs contain all the above-mentioned genes, and they neither showed significant changes in the stemness nor in differentiation markers during 10 days of 2D culture (Figure 4). Based on our results, the content of MSCs-derived MVs did not reflect the autonomous differentiation of MSCs during 10-day 2D culture. Therefore, not only MSCs-derived MVs preserved somewhat their stemness contents but also MSCs maintained proliferative during 10-day 2D culture. Our finding was in accordance with previous studies confirming the regenerative effects of MSCs-derived MVs. Taken together, 2D culture had no significant effect on the biological state of MSCs which strengthened their applications in cell therapies. On the other hand, this research proposed new application of stem cell-derived MVs as therapeutic mediators. Further studies are needed to identify the protein component of MVs to give some benefits for biomimetic approaches. Abbreviations iPSCs induced Pluripotent Stem Cells MSCs Mesenchymal Stem Cells ESCs Embryonic Stem Cells MVs Microvesicles TEM Transmission Electron Microscopy DLS Dynamic Light Scattering bFGF basic Fibroblast Growth Factor PI Propidium Iodide ALP Alkaline Phosphatase Declarations Authorship contribution statement: Conceptualizing the study, methodology, and drafting and revising the manuscript, B. Bakhshandeh. ; experimental works, collecting the data and preparing the results, F. Foroughi Fard. ; methodology, analysis, and drafting and revising the manuscript, Z. Jahanafrooz. All authors read and approved the final manuscript. Conflict of interest: The authors declare no conflicts of interest. Funding statement: This research received no specific grant from any funding agency. Acknowledgment : We appreciate Dr. Ameneh Rezayof and Dr. Bahman Zeynali for their kind contributions. References De Luca M et al (2019) Advances in stem cell research and therapeutic development. 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J Biomed Mater Res A 106(5):1200–1210 Jiang X et al (2018), The role of Sox9 in collagen hydrogel-mediated chondrogenic differentiation of adult mesenchymal stem cells (MSCs) . Biomater Sci 6(6):1556–1568 Supplementary Files GraphicalAbstract.jpg supplementarytable1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Returned Without Review 11 Jul, 2023 Reviewers agreed at journal 22 May, 2023 Reviewers invited by journal 25 Apr, 2023 Editor assigned by journal 21 Apr, 2023 First submitted to journal 20 Apr, 2023 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-2842807","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":194946920,"identity":"58384ed2-ddad-4b6d-a166-0e1acf0f21eb","order_by":0,"name":"Fatemeh Foroughi Fard","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"Foroughi","lastName":"Fard","suffix":""},{"id":194946921,"identity":"0faf6fe6-6e10-4c6a-8810-0bb932064c5b","order_by":1,"name":"Behnaz Bakhshandeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACAwYeBsbGBoYEEJuZoQIqnEC8ljMMEiRqYWyDasEHzBl4D36cuaMuj39288bHhfMO1zGwH37A8HAPbi2WDXzJkhvPHC6WuHOs2HjmtsMSDDxpBgwJz/A47ACPgeTDtgOJDTdyzKR5QVoYcoB+OYBXi/HPh211ifNv5Jj/5p0D1ML/hqAWM8mNbcyJG4C2MPM2ALVIELDFspkvzXJm2+HEjTfSiqV5jqVLtkk8MziAT4s5e+/hm71Ah827kbzxM0+NNT8/f/LDhz/waGFgRhdgA2J8GkbBKBgFo2AUEAEARjJT6sbwlgcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1252-6088","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Behnaz","middleName":"","lastName":"Bakhshandeh","suffix":""},{"id":194946922,"identity":"d0b3069c-f384-4267-ad4b-eb1d88bad76f","order_by":2,"name":"Zohreh Jahanafrooz","email":"","orcid":"","institution":"University of Maragheh","correspondingAuthor":false,"prefix":"","firstName":"Zohreh","middleName":"","lastName":"Jahanafrooz","suffix":""}],"badges":[],"createdAt":"2023-04-20 21:09:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2842807/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2842807/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36426268,"identity":"313cf790-0110-48f8-b70b-9f83cac57a8b","added_by":"auto","created_at":"2023-04-28 13:26:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":380602,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristic of MSCs. (A): Osteogenic differentiation of MSCs indicated by Alizarin staining of calcium oxalates. (B): Chondrogenic differentiation of MSCs indicated by Alcian Blue dye identify of hyaluronic acid (C): Adipogenic differentiation of MSCs indicated by intracellular staining using Oil Red-O. (D): Flow cytometry analysis of cell surface CD markers present on MSCs (positive for CD44 and CD29 while negative for CD34 and CD45).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/302735064e432d83e1e1be11.jpeg"},{"id":36426263,"identity":"67ad4066-b258-46c7-8bc5-9f610b7840a3","added_by":"auto","created_at":"2023-04-28 13:26:48","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49447,"visible":true,"origin":"","legend":"\u003cp\u003eThe rate of apoptosis and necrosis in MSCs characterized by Annexin V/PI dual staining flow cytometry on days 1 and day 10 in 2D culture. The percentage of viable, early apoptotic, late apoptosis, and necrotic cells on days 1, day 10, day10/day1 using a bar chart. Data are presented as means ± SD of at least three independent experiments. Data presents no significance in any group. *P\u0026lt;0.05 was considered as a significant difference compared to the control (untreated group).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/22fd53ae1fad19765cac6771.jpeg"},{"id":36426264,"identity":"49f82483-5086-41b5-a99c-5d013980aedc","added_by":"auto","created_at":"2023-04-28 13:26:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":142656,"visible":true,"origin":"","legend":"\u003cp\u003eMVs characterization; (A): The population homogeneity and size range of isolated MVs obtained by DLS. (B): Round shape of a single isolated MV demonstrated by TEM.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/92566b68791b7c84a60a2f96.jpeg"},{"id":36426267,"identity":"a421eb7d-84cf-4476-9f62-c22d890ef571","added_by":"auto","created_at":"2023-04-28 13:26:48","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47161,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluating real-time content changes of ALP, OCT4, SOX9, Bglap, and RunX2 mRNAs inside the MVs derived from MSCs. Expression of ALP and OCT4 mRNAs was decreased while expression of SOX9, Bglap, and RunX2 mRNAs were increased inside the secreted MVs after day 10. Data are presented as means ± SD of at least three independent experiments. There was no significant difference compared to the control (day 1).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/7696beeb08997e33ee1b96aa.jpeg"},{"id":36427164,"identity":"fd8f3be9-28cb-4ec3-b856-f5e4f558f0ce","added_by":"auto","created_at":"2023-04-28 13:34:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":619646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/a4b50316-c1f2-4288-8acc-c7fa1d6fe084.pdf"},{"id":36426266,"identity":"4c6941c0-3e7a-45ef-9de4-bd3a3f038453","added_by":"auto","created_at":"2023-04-28 13:26:48","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":313837,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/13044a7387e6ef5a29e3928e.jpg"},{"id":36427163,"identity":"76c86d08-3998-495d-8f9b-64e9d7ed1de9","added_by":"auto","created_at":"2023-04-28 13:34:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16790,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2842807/v1/88a6957547d959d9fea14cfc.docx"}],"financialInterests":"","formattedTitle":"The effect of 2D culture on stemness-related transcriptome in shed microvesicles of mesenchymal stem cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStem cells have self-renewal and differentiation capacities, which make them promising candidates for cell therapy. Embryonic stem cells (ESCs), MSCs, hematopoietic stem cells, and induced pluripotent stem cells (iPSCs) are major categories of stem cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. MSCs are found in the most fetus and adult tissues, which are easily available compared to other stem cell types. MSCs have immunomodulatory properties with potential differentiation capacity into diverse cell types (i.e., both mesenchymal and ecto/endodermal lineages). The ability to migrate into injured tissues and direct initiation of tissue repair makes MSCs more favorable in regenerative medicine [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. MSCs have a tropism to tumor tissue and can be used as a cell-based anticancer drug [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Releasing of vesicles, including MVs, exosomes, and apoptotic bodies is a conserved process and a common way of cell-to-cell communication and signaling with tissue-specific contents in almost all cell types [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Exosomes are released by cells upon fusion of multi-vesicular bodies with the plasma membrane while MVs are directly bud from the plasma membrane and transport protein, lipids, mRNA, miRNA, and membrane receptors among the cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, many researches have been focused on the properties of MVs such as small size (i.e., less than 1\u0026micro;m), high flexibility, capability to cross the biological barrier, and protecting capacity of their cargo, to acquire them in therapeutic application such as drug delivery, favorable differentiation and reshaping the injured tissues. MVs derived from stem cells are promising mediators in regenerative medicine. Stem cell-derived MVs therapy is a type of stem cell-based therapies, which provides a safer treatment than whole stem cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mohammadi et al. confirmed the immunomodulatory effects of human MSCs-derived MVs, and the role of MVs as an alternative for MSCs therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent successful translations of stem cell-based therapies to patients have enriched the idea that such strategies may one day become a treatment in regenerative medicine [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, stemness maintenance during \u003cem\u003ein vitro\u003c/em\u003e culture is a key issue for stem cell-based applications in cell therapies. Actually, \u003cem\u003ein vitro\u003c/em\u003e condition and especially 2D culture cannot mimic the natural 3D situation of the body including biochemicals (such as growth factors, cytokines, extracellular components, and microbial-derived compounds) and biophysicals (such as surface topographic textures and mechanical cues) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, repetitive \u003cem\u003ein vitro\u003c/em\u003e cell passages accelerate MSCs senescence leading to decreased stemness state [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, many studies have evaluated the ways to preserve the stemness capacity of stem cells during \u003cem\u003ein vitro\u003c/em\u003e cultures [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Different strategies such as organoid culture, genetic manipulation, optimized cell culture methods, and utilization of biochemical and biophysical signals have been applied for this purpose [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In spite of undesirable effects of 2D culture, due to cost beneficially and simplicity, 2D culture is a well-established and widely used method compared to 3D culture. On the other hand, the developing applications of MVs have prompted us to evaluate the possible changes of MSCs-derived MVs content during 2D culture. Gene expression is a valuable molecular marker associated with stemness characteristic of MSCs. Overexpression of genes involved in embryonic-like phenotype along with downregulation of differentiation related genes can confer the maintenance of stemness state of MSCs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, herein the transcriptional alterations of some stem cell-associated markers namely OCT4 and ALP as well as some differentiation related genes such as SOX9, RunX2, and Bglap genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] inside the secreted MVs were evaluated during 2D culture of MSCs. Overall, the impact of 2D culture on stemness characteristics of MSCs was investigated through focusing on the mRNA content of the secreted MVs.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell isolation and culture\u003c/h2\u003e \u003cp\u003eWe isolated MSCs from the bone marrow of 20-day-old albino female Norway rats, which were obtained from the animal house of University of Tehran. All experimental protocols were performed under ethics approval obtained by the Iran Animal Care Committee. Also, experiments were done in accordance with the approved protocols, institutional guidelines for the care and use of laboratory animals and ARRIVE recommendations. Bone marrow cells were flushed out of the cavity of femurs and tibias, then cultured in DMEM medium (Gibco\u0026trade;) supplemented with 20% FBS (Gibco\u0026trade;) and 1% penicillin/streptomycin (pen/strep) antibiotics (Gibco\u0026trade;) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e under 90\u0026ndash;95% humidity. We removed non-adherent cells 2 and 5 days after primary culture and changed the medium every other day until cells reached 90% confluence. The adherent MSCs were detached by trypsin, reseeded as new cultures, and expanded for further studies. The isolated MSCs were characterized by flow cytometry analyses of CD44, CD29, CD45 and CD34 markers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Differentiation protocols\u003c/h2\u003e \u003cp\u003eTo evaluate the lineage commitment of isolated cells, osteogenic, chondrogenic, and adipogenic differentiation studies were carried out. For osteogenic differentiation, cells were incubated in osteogenic medium (DMEM, 7\u0026ndash;10 mM dexamethasone, 10% FBS, 50 \u0026micro;g/ml ascorbic acid biphosphate, 10 mM \u0026szlig;-glycerol phosphate) for 14 days. To investigate calcium mineralization in osteogenic differentiation, cultured cells were treated with 1% Alizarin Red (Sigma-Aldrich) in pH 4.1 for 10 min at room temperature and visualized under a light microscope.\u003c/p\u003e \u003cp\u003eFor adipogenic induction, the growth medium was supplemented with 107 mM dexamethasone, 0.5 mM IBMX, 0.2 mM indomethacin, 66 nM Insulin for 21 days. Then the cells were stained by Oil Red (Sigma-Aldrich) to detect the accumulated oil droplets.\u003c/p\u003e \u003cp\u003eChondrogenic differentiation was induced by 50 \u0026micro;g/mL ascorbic acid bisphosphate, 250 nM dexamethasone, 200 ng/20 mL basic fibroblast growth factor (bFGF), 1% ITS (6.25 mg/mL insulin, 6.25 mg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid), and 10 ng/mL transforming growth factor-beta (Sigma-Aldrich). To identify the presence of hyaluronic acid in the chondrogenic differentiated cells Alcian Blue dye (Sigma-Aldrich) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell death analysis by Annexin V and propidium iodide (PI) flow cytometry\u003c/h2\u003e \u003cp\u003e To investigate the trends of cell apoptosis and necrosis upon 2D culture, PI and Annexin V-FITC (EXBIO, Vestec, Czech Republic) were utilized for double-staining of MSCs, followed by flow cytometry analysis in day 1 and day 10 of culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Isolation of MVs\u003c/h2\u003e \u003cp\u003eCell cultures were replaced with the serum-free medium about 8 hours before centrifugation to minimize the FBS albumin interference. The supernatants were collected and cell-free supernatants were obtained by 2,000-rpm centrifugation for 10 minutes at 4\u0026deg;C. Apoptotic bodies and cell debris were excluded by 10,000 g centrifugation for 20 minutes at 4\u0026deg;C. MVs pallets were collected after 60,000 g centrifugation at 4\u0026deg;C for one hour twice. The pellets were used freshly. To indirect estimation of the concentration of isolated MVs, from protein concentration, we applied the Bradford assay. In this assay, protein molecules bind to Coomassie dye under acidic conditions resulting in a color change from brown to blue. Absorbance is measured at 595 nm following a short room temperature incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Characterization of MVs\u003c/h2\u003e \u003cp\u003eTo determine the size and morphology of MVs, isolated MVs were stained with 2% uranyl acetate on formvar-carbon-coated grids. After drying, transmission images were provided by placing the grid in an electron microscope (Philips).\u003c/p\u003e \u003cp\u003eDLS technique was used to determine the size range and homogeneity of the isolated MVs by re-suspending the MVs pellet in PBS and analyzing by Malvern instrument (Malvern).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Quantitative transcriptional evaluation\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from MSC-derived MVs on days 1 and 10 of 2D culture using RNX Plus reagent (Cinnagen). Then, reverse transcription of mRNAs was performed using a cDNA synthesized kit according to the manufacturer's instructions (Cinnagen). The real-time PCR reactions were conducted utilizing the SYBR Green master mix (Takara) in Rotor-Gene 6000 Real-Time Thermal Cycler (Corbett Research, Australia). The primer sequences for β2m (housekeeping gene), Alkaline phosphatase (ALP), OCT4, SOX9, Osteocalcin (or Bglap), and RunX2 are provided in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Relative Expression Software Tool (REST 2009, Corbett Research, Australia) was used to measure relative transcriptions through the 2\u003csup\u003e(\u0026minus;∆∆Ct)\u003c/sup\u003e approach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll tests were performed at least in triplicate. Data from two groups were compared by the student\u0026rsquo;s two-tailed t-test. The outcomes were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A p-value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characteristics of the cells\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (A, B, C), red calcium deposition is an indication of osteogenic differentiation while secreted hyaluronic acid is an indication of cartilaginous differentiation. Obvious fat bright red droplets represent the differentiation of MSCs into adipocyte cells. Isolated morphology of MSCs were fibroblastic and spindle-shaped. Analyses of their surface markers by flow cytometry confirmed expression of CD44 and CD29 while these cells did not express CD45 and CD34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Cell viability assessment during 2D culture\u003c/h2\u003e \u003cp\u003eFlow cytometry analyses of annexin V and PI were performed to evaluate the effects of 2D culture on MSCs viability and the type of cell death. There was no significant difference in live, early apoptotic, late apoptotic, and necrotic cell numbers between day 1 and day 10 of the culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characteristics of isolated MVs\u003c/h2\u003e \u003cp\u003e According to Bradford assay, the average protein contents of MVs on day 1 and day 10 were 9.5 5 \u0026micro;g/\u0026micro;L and 18.5 \u0026micro;g/\u0026micro;L, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the average diameter of MVs was less than 1 \u0026micro;m with a round shape, confirming the efficacy of the protocol for the isolation and purification of MVs. DLS analysis showed that the shedding MVs were distributed in the range from 100 nm to 1000 nm in diameter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Quantitative evaluation of the mRNA content of the MVs during 2D culture\u003c/h2\u003e \u003cp\u003eGene expression analysis by quantitative PCR showed that mRNAs contents of MSCs-derived MVs on day 10 had no significant changes compared to day 1. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the patterns of transcriptional changes in the ALP, OCT4, SOX9, Osteocalcin, and RunX2 genes were different. While ALP and OCT4 mRNAs slightly decreased on day 10, SOX9, Bglap, and RunX2 mRNAs showed the opposite pattern inside the MVs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion and future remarks","content":"\u003cp\u003eOver the past years, the efficacy of MSCs, as the main players in cell therapy, has been reported in various clinical trials\u0026nbsp;[21]. The application of MSCs in medicine\u0026nbsp;is\u0026nbsp;a rapidly evolving approach and\u0026nbsp;is\u0026nbsp;a promising option for regenerative medicine\u0026nbsp;[8, 22, 23]. So, increasing demand for MSCs and their derivations as cell-based drugs in the market is anticipated.\u0026nbsp;However, some drawbacks, such as the pro-tumorigenic effect of implanted MSCs, short-time viability after injection, reaching low portions of MSCs to damaged tissue after systemic administration, the immunogenicity of the differentiated MSCs, induction of allo-antibodies in repeated administration of MSCs, and induction of antibodies\u0026nbsp;against fetal bovine serum (FBS) used in the MSC culture medium have hindered this issue\u0026nbsp;[24]. In this regard, the application of MSCs derivations such as shed MVs can be considered as a safer approach. Cha et al. showed that human MSCs secret remarkable MVs, and confirmed the\u0026nbsp;therapeutic capacity of MSC-derived MVs\u0026nbsp;[11].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne of the major challenges in MSC therapy is\u0026nbsp;a possible alteration in\u0026nbsp;stemness potential of MSCs during in vitro culture and proliferation\u0026nbsp;[25]. During MSCs in vitro culture, autonomous osteoblastic self-differentiation was reported\u0026nbsp;[26, 27]. Since MVs carry various cargos such as mRNA, proteins, miRNA, bioactive lipids, and signaling nucleotides, investigation of shed MVs could reflect the situation of the origin cells\u0026nbsp;[28, 29]. Herein, the alterations in stemness potential of MSCs during a 10-day 2D culture was investigated both from cell proliferative status and their transcriptome in secreted MVs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEfficient expansion of the MSCs while avoiding any culture-related phenotypic changes and cell death, is an important challenge in MSCs therapy\u0026nbsp;[30, 31]. In our study, apoptotic cell death of MSCs during a 10-day 2D culture was negligible (Figure 2).\u0026nbsp;A neglectable decrease in the percentage of viable cells during cell culture has been reported in many studies in accordance with our study\u0026nbsp;[31].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor indirect inspection of 10-day cultured MSCs, their shed MVs were isolated. Characterization of isolated MVs from MSCs, including shape, diameter, and size distribution (Figure 3) was in accordance with previous studies\u0026nbsp;[32, 33]. There are few studies about the mRNA, miRNA, and protein contents of MVs. Xie, et al. reported regulation of hematopoiesis by miRNAs in MSCs-derived MVs applied in ex vivo expansion of cord blood mononuclear cells\u0026nbsp;[33]. In another study, MSCs-derived MVs enhanced the survival of renal cells both in vitro and in vivo\u0026nbsp;[32]. Herein some stemness markers such as OCT4 and ALP\u0026nbsp;[34]\u0026nbsp;were evaluated. In addition, some differentiation markers such\u0026nbsp;as\u0026nbsp;Bglap and RunX2 (osteogenic potential markers\u0026nbsp;[35, 36]) and\u0026nbsp;SOX9\u0026nbsp;(a marker of chondrogenic differentiation\u0026nbsp;[37]) were also investigated\u0026nbsp;[35-37]. Based on our findings, MSCs-derived MVs contain all the above-mentioned genes, and they neither showed significant changes in the stemness nor in differentiation markers during 10 days of 2D culture (Figure 4). Based on our results, the content of MSCs-derived MVs did not reflect the autonomous differentiation of MSCs during 10-day 2D culture. Therefore, not only\u0026nbsp;MSCs-derived MVs preserved somewhat their stemness contents but also MSCs maintained proliferative during 10-day 2D culture. Our finding was in accordance with previous studies confirming the regenerative effects of MSCs-derived MVs.\u003c/p\u003e\n\u003cp\u003eTaken together, 2D culture had no significant effect on the biological state of MSCs which strengthened their applications in cell therapies. On the other hand, this research proposed new application of stem cell-derived MVs as therapeutic mediators. Further studies are needed to identify the protein component of MVs to give some benefits for biomimetic approaches.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eiPSCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003einduced Pluripotent Stem Cells\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMSCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eMesenchymal Stem Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eESCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eEmbryonic Stem Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMVs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eMicrovesicles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eTransmission Electron Microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDLS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eDynamic Light Scattering\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ebFGF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003ebasic Fibroblast Growth Factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003ePropidium Iodide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eALP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.71428571428571%\" valign=\"top\"\u003e\n \u003cp\u003eAlkaline Phosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement:\u003c/strong\u003e Conceptualizing the study, methodology, and drafting and revising the manuscript, \u003cstrong\u003eB. Bakhshandeh.\u003c/strong\u003e; experimental works, collecting the data and preparing the results, \u003cstrong\u003eF.\u003c/strong\u003e\u003cstrong\u003eForoughi Fard.\u003c/strong\u003e; methodology,\u0026nbsp;analysis,\u0026nbsp;and drafting and revising the manuscript, \u003cstrong\u003eZ. Jahanafrooz.\u003c/strong\u003e All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement:\u003c/strong\u003e This research received no specific grant from any funding agency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e: We appreciate Dr. Ameneh Rezayof and Dr. Bahman Zeynali for their kind contributions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eDe Luca M et al (2019) Advances in stem cell research and therapeutic development. Nat Cell Biol 21(7):801\u0026ndash;811\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHafizi M et al (2012) Exploring the enkephalinergic differentiation potential in adult stem cells for cell therapy and drug screening implications. 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Nat Rev Mater 7(6):424\u0026ndash;424\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKouroupis D, Correa D (2021) Increased mesenchymal stem cell functionalization in three-dimensional manufacturing settings for enhanced therapeutic applications. Front Bioeng Biotechnol 9:621748\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eStahl PD, Raposo G (2019) Extracellular Vesicles: Exosomes and Microvesicles, Integrators of Homeostasis. Physiology 34(3):169\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRen S et al (2019) Microvesicles from human adipose stem cells promote wound healing by optimizing cellular functions via AKT and ERK signaling pathways. Stem Cell Res Ther 10(1):1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGarvican ER et al (2014) Viability of equine mesenchymal stem cells during transport and implantation. Stem Cell Res Ther 5(4):1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMohyeddin Bonab M et al (2021) A novel method for maintaining the stability of freshly cultured Mesenchymal stem cells in clinical grade injection ready state without cryopreservation. Transl Med Commun 6(1):1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKosanović M et al (2022) Extracellular Vesicles and Acute Kidney Injury: Potential Therapeutic Avenue for Renal Repair and Regeneration. Int J Mol Sci 23(7):3792\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXie H et al (2016) \u003cem\u003eMesenchymal stem cell-derived microvesicles support ex vivo expansion of cord blood-derived CD34 + cells\u003c/em\u003e.Stem Cells Int., : p.2016\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKaushik G et al (2021) Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis. Oncogene 40(4):848\u0026ndash;862\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYang JX et al (2020) Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2. Cell Signal 70:109504\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOftadeh MO et al (2018) Sequential application of mineralized electroconductive scaffold and electrical stimulation for efficient osteogenesis. J Biomed Mater Res A 106(5):1200\u0026ndash;1210\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJiang X et al (2018), \u003cem\u003eThe role of Sox9 in collagen hydrogel-mediated chondrogenic differentiation of adult mesenchymal stem cells (MSCs)\u003c/em\u003e. Biomater Sci 6(6):1556\u0026ndash;1568\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microvesicles, Mesenchymal stem cells, Apoptosis, Stemness potential, 2D culture","lastPublishedDoi":"10.21203/rs.3.rs-2842807/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2842807/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOver the past years, the applications of mesenchymal stem cells (MSCs) grow rapidly in medicine as a promising option for regenerative medicine. So, increasing demands for MSCs and their derivations as cell-based drugs in the market are anticipated. One of the major challenges in MSC therapy is possible alterations in the stemness potential of MSCs during in vitro culture and proliferation. Microvesicles (MVs) reflect the transcriptional status of cells of origin. This study aimed to investigate the impact of 2D culture on stemness characteristics of MSCs by focusing on transcriptome in shed MVs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods and Results\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMVs were isolated from rat bone marrow MSCs using ultracentrifugation. Transmission electron microscopy and dynamic light scattering were applied to characterizing MVs. Then, some markers related to stemness and differentiation were evaluated in shed MVs during 10-day culture. Meanwhile the cell proliferation and apoptosis were assessed in cultured MSCs. Based on our results, not only MSCs-derived MVs preserved somewhat their stemness contents but also MSCs maintained proliferative during 10-day 2D culture.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTaken together, 2D culture had no significant effect on the biological state of MSCs which strengthened their applications in cell therapies. Further studies are needed to identify the protein component of MVs to give some benefits for biomimetic approaches.\u003c/p\u003e","manuscriptTitle":"The effect of 2D culture on stemness-related transcriptome in shed microvesicles of mesenchymal stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-28 13:26:43","doi":"10.21203/rs.3.rs-2842807/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Returned Without Review","date":"2023-07-11T12:07:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-05-22T12:37:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-25T11:42:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-21T14:58:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-04-20T17:09:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1844894d-c2c4-405d-8157-29a0ab26dd1f","owner":[],"postedDate":"April 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-04-28T13:26:43+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-28 13:26:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2842807","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2842807","identity":"rs-2842807","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}