Human multipotent mesenchymal stromal cells cytokine priming promotes RAB27B-regulated secretion of small extracellular vesicles with immunomodulatory cargo

Abstract

Background The 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. Methods We 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). Results Cytokine 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. Conclusions Cytokine 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.

Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

“…Overall, the interaction of these surface markers on exosomes with monocytes may improve the capacity of the educated monocytes to drive the tissue repair in ARS. It is important to note that TLR-4 stimulation by LPS is not the only method to prime MSCs and generate exosomes, as TNFα [ 20 – 22 ], IFNγ [ 22 , 23 ], and IL-1β [ 24 , 25 ] have also been shown as effective molecules for priming MSCs. A head-to-head comparison of MSC priming methodologies needs to be conducted.…”

The Unique Immunomodulatory Properties of MSC-Derived Exosomes in Organ Transplantation

“…Bruno et al ( 37 ) revealed that exosomes play an important role in the paracrine effect of MSCs. MSC-derived exosomes with cytokines and growth factors, lipid signals, mRNA, and miRNA regulatory biomolecules have a biological function or information transfer function ( 38 , 39 ). They have similar functions to MSCs, including the promotion of tissue repair, immunosuppression, and neuroprotection.…”

“…However, methods for obtaining exosomes with high- quantity and high-purity remain a focus of research. Cytokine priming upregulated RAB27B siRNA in AD-MSCs, increasing their secretion of exosomes, which is a useful strategy for harvesting anti-inflammatory MSC-EVs for clinical applications ( 39 ). As mentioned earlier, gene editing is used to overexpress therapeutically effective molecules (such as mRNA, miRNA, and proteins) in MSCs to produce characteristic MSC-derived exosomes for different diseases.…”

Adipose-Derived Mesenchymal Stromal Cells Treated with Interleukin 1 Beta Produced Chondro-Protective Vesicles Able to Fast Penetrate in Cartilage

“…In basal conditions, ASC released extracellular vesicles (EVs) with a preponderance of microRNAs (miRNAs) involved in OA cartilage and synovium protection (40%) over less than 10% of those involved in degenerative processes [ 5 ]. In addition, the EV cargo can be further modulated by preconditioning the mesenchymal stem cells (MSCs) with stimuli resembling the in vivo pathological environment [ 6 , 7 , 8 ]. ASCs inflammatory priming with interferon γ (IFNγ) determined a production of a 34.7% chondro-protective versus a 13.3% chondro-destructive miRNAs and a secretion of 26.2% versus 4.5% of M2- versus M1-macrophages-related miRNAs [ 9 ].…”