Preconditioning in an Inflammatory Milieu Augments the Immunotherapeutic Function of Mesenchymal Stromal Cells

Abstract

Multipotent mesenchymal stromal cells (MSCs) have emerged as potent therapeutic agents for multiple indications. However, recent evidence indicates that MSC function is compromised in the physiological post-injury milieu. In this study, bone marrow (BM)- and adipose-derived (AD)-MSCs were preconditioned in hypoxia with or without inflammatory mediators to potentiate their immunotherapeutic function in preparation for in vivo delivery. Human MSCs were cultured for 48 h in either normoxia (21% O2) or hypoxia (2% O2) with or without the addition of Cytomix, thus creating 4 groups: (1) normoxia (21%); (2) Cytomix-normoxia (+21%); (3) hypoxia (2%); and (4) Cytomix-hypoxia (+2%). The 4 MSC groups were subjected to comprehensive evaluation of their characteristics and function. Preconditioning did not alter common MSC surface markers; nonetheless, Cytomix treatment triggered an increase in tissue factor (TF) expression. Moreover, the BM-MSCs and AD-MSCs from the +2% group were not able to differentiate to chondrocytes and osteoblasts, respectively. Following Cytomix preconditioning, the metabolism of MSCs was significantly increased while viability was decreased in AD-MSCs, but not in BM-MSCs. MSCs from both tissues showed a significant upregulation of key anti-inflammatory genes, increased secretion of IL-1 receptor antagonist (RA), and enhanced suppression of T-cell proliferation following the Cytomix treatment. Similarly, following a lipopolysaccharide challenge, the Cytomix-treated MSCs suppressed TNF-α secretion, while promoting the production of IL-10 and IL-1RA. These preconditioning approaches facilitate the production of MSCs with robust anti-inflammatory properties. AD-MSCs preconditioned with Cytomix under normoxia appear to be the most promising therapeutic candidates; however, safety concerns, such as thrombogenic disposition of cells due to TF expression, should be carefully considered prior to clinical translation.

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

“…In one experimental setting, UCMSCs were stimulated with pro-inflammatory cytokines (hereafter referred to as 'primed') for 48 h when they reached 80% confluence. They were treated with an inflammatory cocktail containing 5 ng/mL TNF-α, 2.5n g/mL IFN-γ and 2.5 ng/mL IL-1β (Peprotech, London, UK) [11,14,15]. Figure 1 outlines the experimental plan and culture conditions of UCMSCs.…”

“…The MSCs secretome is variable, and the conditions MSCs are exposed to can change their functional properties [10]. For example, MSC-EVs from cells primed with pro-inflammatory cytokines (Tumor necrosis alpha (TNF-α), Interleukin 1 beta (IL-1β) and Interferon gamma (IFN-γ) have shown enhanced immunosuppressive properties against T-cells [11]. Likewise, MSC-EVs derived from cells grown in hypoxia (5% O 2 ) have also shown an enhanced ability to polarise M1 macrophages into M2 macrophages in mice compared to the MSC-EVs derived from normal oxygen conditions [12].…”

Skin Immunomodulation during Regeneration: Emerging New Targets

“…Visceral ADSCs secrete higher quantities of inflammatory cytokines IL-6, IL-8, and TNF-α when compared to the subcutaneous ones [ 165 ]. ADSCs preconditioning with inflammatory or pro-inflammatory cytokines increase their survival and improved their responses to cancer and inflammation [ 32 , 153 , 165 , 166 , 167 ]. This epigenetically modification of the microenvironment of ADSCs might address cell ability to differentiate and migrate, and to restore cellular defects associated to aging.…”

“…Indeed, ADSCs have been reported to regulate inflammation and participate in the phases of wound healing through their exosomes [ 10 , 24 , 25 , 26 , 27 , 28 , 29 ]. Within their microenvironment, ADSCs carried their immune-modulatory and regenerative effects through direct interaction with activated immune cells [ 30 , 31 , 32 ]. This cell network is the key regulator of skin regeneration and repair.…”

SOCS1 Regulates the Immunomodulatory Roles of MSCs on B Cells

“…Studies have shown that the concentration of inflammatory cytokines affects the immunomodulatory effects of MSCs on T cells (23)(24)(25). High concentrations of inflammatory factors can cause MSCs to exertan immunosuppressive effect, while insufficient levels of inflammatory factors can cause MSCs to play an immunological enhancement role (23).…”