BackgroundIn the bone marrow, MSCs reside in a hypoxic milieu (1–5% O2) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O2), a process that has been shown to impair their function. Here, we evaluated the characteristics and function of MSCs cultured under hypoxia and hypothesized that, when compared to normoxia, dedicated hypoxia will augment the functional characteristics of MSCs.MethodsHuman and porcine bone marrow MSCs were obtained from fresh mononuclear cells. The first study evaluated MSC function following both long-term (10 days) and short-term (48 h) hypoxia (1% O2) culture. In our second study, we evaluated the functional characteristics of MSC cultured under short-term 2% and 5% hypoxia. MSCs were evaluated for their metabolic activity, proliferation, viability, clonogenicity, gene expression, and secretory capacity.ResultsIn long-term culture, common MSC surface marker expression (CD44 and CD105) dropped under hypoxia. Additionally, in long-term culture, MSCs proliferated significantly slower and provided lower yields under hypoxia. Conversely, in short-term culture, MSCs proliferated significantly faster under hypoxia. In both long-term and short-term cultures, MSC metabolic activity was significantly higher under hypoxia. Furthermore, MSCs cultured under hypoxia had upregulated expression of VEGF with concomitant downregulation of HMGB1 and the apoptotic genes BCL-2 and CASP3. Finally, in both hypoxia cultures, the pro-inflammatory cytokine, IL-8, was suppressed, while levels of the anti-inflammatories, IL-1ra and GM-CSF, were elevated in short-term hypoxia only.ConclusionsIn this study, we demonstrate that hypoxia augments the therapeutic characteristics of both porcine and human MSCs. Yet, short-term 2% hypoxia offers the greatest benefit overall, exemplified by the increase in proliferation, self-renewing capacity, and modulation of key genes and the inflammatory milieu as compared to normoxia. These data are important for generating robust MSCs with augmented function for clinical applications.
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.
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