HIF-1α and Pro-Inflammatory Signaling Improves the Immunomodulatory Activity of MSC-Derived Extracellular Vesicles

Abstract

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.

TNF-α and IFN-γ Participate in Improving the Immunoregulatory Capacity of Mesenchymal Stem/Stromal Cells: Importance of Cell–Cell Contact and Extracellular Vesicles

“…Similar results have been seen in MSCs whose senescence is induced by radiation [170], oxidative stress [171], or exposure to pro-inflammatory cytokines [172]. Because such alterations can affect the therapeutic efficacy of these cells, it has currently been proposed to remove senescent cells before being administered to patients [173] or to establish non-senescent MSC lines [120].…”

“…Stimulation of MSCs with proinflammatory cytokines increases the amount of EVs released by these cells [117][118][119][120], and changes its content which influences their immunoregulatory capacity. In addition, EVs released by resting or activated BM-MSCs do not express MHC-I or MHC-II [98].…”

“…Likewise, the stimulation of MSCs derived from dental pulp (DP-MSCs) with IFN-γ, TNFα, IL-1β increases the release of EV-mix enriched in PD-L1, IDO, and COX2, which decrease the proliferation of CD4+ and CD8+ T lymphocytes. Moreover, the administration of these structures in a delayed-type hypersensitivity mouse model, improves the integrity of the tissues with a higher presence of M2 cells, and decreases the infiltration of CD45+ cells and M1 macrophages [120]. Similar results have been obtained with EV-mix released by murine BM-MSCs exposed to IL-6, TNF-α, and IL-1β, its administration in a murine model of colitis reduces the clinical-pathological signs of the disease.…”

miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia

“…To overcome this problem, we previously immortalized dental pulp-derived primary MSC cultures by overexpressing human telomerase enzyme (termed MSC-T) using a lentiviral vector that provided resistance to hygromycin and increased telomerase activity. MSC-T exhibit prolonged lifespan and maintain cell division long after equivalent unmodified MSCs reach replicative senescence ( Gómez-Ferrer et al, 2021 ). We harvested EVs from conditioned media of MSC-T cells using ultracentrifugation as described ( Gonzalez-King et al, 2017 ).…”

“…The immortal MSC-TERT line was used as previously described ( Gómez-Ferrer et al, 2021 ). Donor cells were cultured in DMEM-low glucose supplemented with 1% penicillin/streptomycin and 10% FBS (Lonza, Basel, Switzerland).…”