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Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles.

ABSTRACT: Umbilical cord mesenchymal stromal cells (UCMSCs) have shown an ability to modulate the immune system through the secretion of paracrine mediators, such as extracellular vesicles (EVs). However, the culture conditions that UCMSCs are grown in can alter their secretome and thereby affect their immunomodulatory potential. UCMSCs are commonly cultured at 21% O2 in vitro, but recent research is exploring their growth at lower oxygen conditions to emulate circulating oxygen levels in vivo. Additionally, a pro-inflammatory culture environment is known to enhance UCMSC anti-inflammatory potential. Therefore, this paper examined EVs from UCMSCs grown in normal oxygen (21% O2), low oxygen (5% O2) and pro-inflammatory conditions to see the impact of culture conditions on the EV profile. EVs were isolated from UCMSC conditioned media and characterised based on size, morphology and surface marker expression. EV protein cargo was analysed using a proximity-based extension assay. Results showed that EVs had a similar size and morphology. Differences were found in EV protein cargo, with pro-inflammatory primed EVs showing an increase in proteins associated with chemotaxis and angiogenesis. This showed that the UCMSC culture environment could alter the EV protein profile and might have downstream implications for their functions in immunomodulation.

SUBMITTER: Hyland M

PROVIDER: S-EPMC7140705 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles.

Hyland Mairead M Mennan Claire C Wilson Emma E Clayton Aled A Kehoe Oksana O

Cells 20200316 3

Umbilical cord mesenchymal stromal cells (UCMSCs) have shown an ability to modulate the immune system through the secretion of paracrine mediators, such as extracellular vesicles (EVs). However, the culture conditions that UCMSCs are grown in can alter their secretome and thereby affect their immunomodulatory potential. UCMSCs are commonly cultured at 21% O<sub>2</sub> in vitro, but recent research is exploring their growth at lower oxygen conditions to emulate circulating oxygen levels in vivo. ...[more]

PMID: 32188006

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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-γ) and tumor necrosis factor alpha (TNF-α) 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.

Project description:Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms. Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated. Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation. Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.

Project description:BackgroundSpinal cord injury (SCI) is an inflammatory condition, and excessive adenosine triphosphate (ATP) is released into the extracellular space, which can be catabolized into adenosine by CD73. Extracellular vesicles have been designed as nano drug carriers in many diseases. However, their impacts on delivery of CD73 after SCI are not yet known. We aimed to construct CD73 modified extracellular vesicles and explore the anti-inflammatory effects after SCI.MethodsCD73 engineered extracellular vesicles (CD73+ hucMSC-EVs) were firstly established, which were derived from human umbilical cord mesenchymal stem cells (hucMSCs) transduced by lentiviral vectors to upregulate the expression of CD73. Effects of CD73+ hucMSC-EVs on hydrolyzing ATP into adenosine were detected. The polarization of M2/M1 was verified by immunofluorescence. Furthermore, A2aR and A2bR inhibitors and A2bR knockdown cells were used to investigate the activated adenosine receptor. Biomarkers of microglia and levels of cAMP/PKA were also detected. Repetitively in vivo study, morphology staining, flow cytometry, cytokine analysis, and ELISA assay, were also applied for verifications.ResultsCD73+ hucMSC-EVs reduced concentration of ATP and promoted the level of adenosine. In vitro experiments, CD73+ hucMSC-EVs increased macrophages/microglia M2:M1 polarization, activated adenosine 2b receptor (A2bR), and then promoted cAMP/PKA signaling pathway. In mice using model of thoracic spinal cord contusion injury, CD73+ hucMSC-EVs improved the functional recovery after SCI through decreasing the content of ATP in cerebrospinal fluid and improving the polarization from M1 to M2 phenotype. Thus, the cascaded pro-inflammatory cytokines were downregulated, such as TNF-α, IL-1β, and IL-6, while the anti-inflammatory cytokines were upregulated, such as IL-10 and IL-4.ConclusionsCD73+ hucMSC-EVs ameliorated inflammation after spinal cord injury by reducing extracellular ATP, promoting A2bR/cAMP/PKA pathway and M2/M1 polarization. CD73+ hucMSC-EVs might be promising nano drugs for clinical application in SCI therapy.

Dataset Information

Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles.

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Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles.

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