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Interferon-? and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles.

ABSTRACT: Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming methods of MSC, interferon-gamma (IFN?) and hypoxia (HYP), could modify their EV miRNA content. Human bone marrow MSCs (BM-MSCs) from five healthy donors were cultured with IFN? or in HYP or in control (CONT) conditions. The conditioned media were collected after 48 h in serum-free condition and EV were isolated by ultracentrifugation. Total RNA was isolated, pools of CONT, IFN, and HYP cDNA were prepared, and a miRNA profiling was performed using RT-qPCR. Then, miRNAs were selected based on their detectability and measured on each individual EV sample. Priming had no effect on EV amount or size distribution. A set of 81 miRNAs was detected in at least one of the pools of EVs. They were measured on each individual sample; 41 miRNAs were detected in all samples. The principal component analysis (PCA) failed to discriminate the groups. HYP induced a significant decrease in EV hsa-miR-34a-3p content and IFN induced a significant increase in five miRNAs (hsa-miR-25-3p, hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-451a, and hsa-miR-665). Taken together, we found only limited alterations in the miRNA landscape of MSC EV with a high inter-individual variability.

SUBMITTER: Peltzer J

PROVIDER: S-EPMC7769832 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Interferon-γ and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles.

Peltzer Juliette J Lund Kyle K Goriot Marie-Emmanuelle ME Grosbot Marion M Lataillade Jean-Jacques JJ Mauduit Philippe P Banzet Sébastien S

Frontiers in cell and developmental biology 20201215

Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming meth ...[more]

PMID: 33384991

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Project description:UnlabelledPreterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells.SignificanceBone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration of living cells. The therapeutic efficacy of systemically administered mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) on hypoxia-ischemia-induced injury was assessed in the preterm ovine brain. Impaired function and structural injury of the fetal brain was improved following global hypoxia-ischemia. A cell-free preparation of MSC-EVs could substitute for the cellular counterpart in the treatment of preterm neonates with hypoxic-ischemic brain injury. This may open new clinical applications for "off-the-shelf" interventions with MSC-EVs.

Project description:Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and ΔCt method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields.

Project description:BackgroundThe paracrine effects of multipotent mesenchymal stromal cells (MSCs) are mediated by their secretome composed by soluble factors (i.e., cytokines, growth factors, hormones) and extracellular vesicles (EVs). EVs promote intercellular communication, and the EV cargoes [e.g., proteins, soluble factors, microRNAs (miRNAs), messenger RNA (mRNA), DNA] reflect the molecular and functional characteristics of their parental cells. MSC-derived EVs (MSC-EVs) are currently evaluated as subcellular therapeutics. A key function of the MSC secretome is its ability to promote immune tolerance (i.e., immunopotency), a property that is enhanced by priming approaches (e.g., cytokines, hypoxia, chemicals) and inversely correlates with the age of the MSC donors. We evaluated mechanisms underlying MSC vesiculation and the effects of inflammation and aging on this process.MethodsWe evaluated the effects of interferon gamma (IFN-?) and tumor necrosis factor alpha (TNF-?) on human adipose-derived MSC: (a) vesiculation (custom RT2 Profiler PCR Array), (b) EV profiles (Nanoparticle Tracking Analysis and Nanoparticle Flow Cytometry), (c) EV cargo (proteomic analysis and Western blot analysis), and (d) immunopotency (standard MSC:CD4 T cell proliferation inhibition assay). We confirmed the role of RAB27B on MSC vesiculation (RAB27B siRNA) and assessed its differential contribution to vesiculation in adult and pediatric MSCs (qPCR).ResultsCytokine priming upregulated RAB27B in adipose-derived MSCs increasing their secretion of exosome-like small EVs (sEVs; <?200?nm) containing two key mediators of immunopotency: A20 and TSG-6. These EVs inhibited T cell proliferation in a dose-dependent manner. RAB27B siRNA inhibited MSC vesiculation. Adipose-derived MSCs isolated from pediatric donors exhibited higher RAB27B expression and secreted more sEVs than adult MSCs.ConclusionsCytokine priming is a useful strategy to harvest anti-inflammatory MSC-sEVs for clinical applications. Of relevance, donor age should be considered in the selection of MSC-sEVs for clinical applications.

Dataset Information

Interferon-? and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles.

Publications

Interferon-γ and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles.

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