Enhanced Therapeutic Potential of Preactivated Mesenchymal Stromal Cells and Their Secretory Factor Stanniocalcin-1 in Models for Acute Injuries and Inflammation

There has been significant interest in the therapeutic potential of the adult stem/progenitor cells from bone marrow called multipotent mesenchymal stromal cells (MSCs). Signals from injured tissues activate MSCs to secrete beneficial factors and contribute to immune/inflammatory modulation and tissue healing. In order to enhance the therapeutic potential of MSCs hanging drop culture method was used to preactivate the cells in vitro and eliminate the lag period required for their activation in vivo. Stanniocalcin-1 (STC-1) is a potent anti-inflammatory and anti-apoptotic protein secreted from activated MSCs and has been considered as a substitute for MSCs in several disease conditions; therefore, the effects of STC-1 were studied on monocyte fate in vitro and in a mouse model of ischemic myocardial injury. Aggregated MSCs in hanging drops were self-activated to produce several therapeutic factors such as anti-inflammatory protein STC-1 and TNFα stimulated gene/protein 6 (TSG-6). MSCs dissociated from spheroids were also smaller than MSCs from standard 2 dimensional cultures, and as a result, larger numbers of them trafficked through the lung of mice after intravenous administration. Notably, spheroid MSCs were more effective than MSCs from standard cultures in suppressing inflammatory responses in a co-culture system with activated macrophages and in a mouse model of peritonitis. The data suggest enhanced therapeutic potential of spheroid MSCs for diseases caused by unresolved inflammation. Treatment with STC-1 reduced expression of CD14, a coreceptor for toll-like receptors, in differentiating monocytes/macrophages and suppressed the inflammatory responses of the cells to endotoxin. Administration of STC-1 also reduced CD14 expression in monocytes stimulated with various danger signals and in hearts of mice after myocardial infarction. These findings may explain the observed decreases in cardiac inflammation following myocardial infarction, and the improvements in ejection fraction and infarct size. The results suggest that STC-1 is a promising therapy to protect the heart and other tissues from ischemic injury

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

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 hours 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

Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells

Abstract Background In 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. Methods Human 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. Results In 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. Conclusions In 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

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Mesenchymal Stem Cells Reconditioned in Their Own Serum Exhibit Augmented Therapeutic Properties in the Setting of Acute Respiratory Distress Syndrome

Abstract Mesenchymal stem cells (MSCs) are a promising form of therapy for acute respiratory distress syndrome (ARDS). The objective of this study was twofold: (a) to characterize cytokine expression in serum from ARDS subjects receiving MSCs and (b) to determine MSC function following “preconditioning” with ARDS serum. In phase I, serum from three cohorts of animals (uninjured [no ARDS, n = 4], injured untreated [n = 5], and injured treated with approximately 6 million per kilogram MSCs [n = 7]) was analyzed for expression of inflammatory mediators. In phase II, the functional properties of bone marrow porcine MSCs were assessed following “preconditioning” with serum from the three cohorts. In phase III, the findings from the previous phases were validated using human bone marrow MSCs (hBM‐MSCs) and lipopolysaccharide (LPS). Serum from injured treated animals had significantly lower levels of interferon‐γ and significantly higher levels of interleukin (IL)‐1 receptor antagonist (IL‐1RA) and IL‐6. Similarly, upon exposure to the injured treated serum ex vivo, the MSCs secreted higher levels of IL‐1RA and IL‐10, dampened the secretion of proinflammatory cytokines, exhibited upregulation of toll‐like receptor 4 (TLR‐4) and vascular endothelial growth factor (VEGF) genes, and triggered a strong immunomodulatory response via prostaglandin E2 (PGE2). hBM‐MSCs demonstrated a similar augmented therapeutic function following reconditioning in a LPS milieu. Administration of MSCs modulated the inflammatory milieu following ARDS. Exposure to ARDS serum ex vivo paralleled the trends seen in vivo, which appear to be mediated, in part, through TLR‐4 and VEGF and PGE2. Reconditioning MSCs in their own serum potentiates their immunotherapeutic function, a technique that can be used in clinical applications. Stem Cells Translational Medicine 2019;8:1092–110