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Background: Mesenchymal stem cells (MSCs) are attractive cell-therapy candidates. Despite their popularity and promise, there is no uniform method of preparation of MSCs. Typically, cells are cryopreserved in liquid nitrogen, thawed, and subsequently administered to a patient with little to no information on their function post-thaw. We hypothesized that a short acclimation period post-thaw will facilitate the recovery of MSC's functional potency., Methods: Human bone-marrow-derived MSCs were divided into 3 groups: FC (fresh cells; from existing culture); TT (thawed + time; acclimated for 24 h post-thaw); and FT (freshly thawed; thawed and immediately used). The 3 groups were analyzed for their cellular and functional potency., Results: Phenotypic analysis demonstrated a decrease in CD44 and CD105 surface markers in FT MSCs, with no change in the other two groups. All MSCs were able to differentiate down the osteogenic and chondrogenic lineages. In FT cells, metabolic activity and apoptosis was significantly increased with concomitant decrease in cell proliferation; clonogenic capacity; and key regenerative genes. Following 24-h acclimation, apoptosis was significantly reduced in TT cells with a concomitant upregulation in angiogenic and anti-inflammatory genes. While all MSCs significantly arrested T-cell proliferation, the TT MSCs were significantly more potent. Similarly, although all MSCs maintained their anti-inflammatory properties, IFN-γ secretion was significantly diminished in FT cells., Conclusions: These data demonstrate that FT MSCs maintain their multipotent differentiation capacity, immunomodulatory function, and anti-inflammatory properties; yet, various aspects of cell characteristics and function are deleteriously affected by cryopreservation. Importantly, a 24-h acclimation period 'reactivates' thawed cells to recover their diminished stem-cell function.

Background: In the bone marrow, MSCs reside in a hypoxic milieu (1-5% O 2 ) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O 2 ), 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% O 2 ) 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.

Background: It is known that, following a physiological insult, bone marrow-derived mesenchymal stem cells (MSCs) mobilize and home to the site of injury. However, the effect of injury on the function of endogenous MSCs is unknown. In this study, MSCs harvested from the bone marrow of swine with or without acute respiratory distress syndrome (ARDS) were assessed for their characteristics and therapeutic function., Methods: MSCs were harvested from three groups of anesthetized and mechanically ventilated swine (n = 3 in each group): 1) no ARDS ('Uninjured' group); 2) ARDS induced via smoke inhalation and 40% burn and treated with inhaled epinephrine ('Injured Treated' group); and 3) ARDS without treatment ('Injured Untreated' group). Cellular evaluation of the three groups included: flow cytometry for MSC markers; colony forming unit-fibroblast (CFU-F) assay; proliferative and metabolic capacity; gene expression using quantitative real-time polymerase chain reaction (qRT-PCR); and a lipopolysaccharide (LPS) challenge, with or without coculture with mononuclear cells (MNCs), for evaluation of their protein secretion profile using Multiplex. Statistical analysis was performed using one- or two-way analysis of variance (ANOVA) with a Tukey's post-test; a p-value less than 0.05 was considered statistically significant., Results: Cells from all groups exhibited nearly 100% expression of MSC surface markers and retained their multidifferentiation capacity. However, the MSCs from the 'Injured Untreated' group generated a significantly higher number of colonies compared with the other two groups (p < 0.0001), indicative of increased clonogenic capacity following ARDS. Following an LPS challenge, the MSCs from the 'Injured Untreated' group exhibited a significant reduction in their proliferative capacity (p = 0.0002), significant downregulation in the expression of high-mobility group box 1 (HMGB1; p < 0.001), Toll-like receptor (TLR)-4 (p < 0.01), and vascular endothelial growth factor (VEGF; p < 0.05) genes, and significantly diminished secretory capacity for the inflammatory mediators interleukin (IL)-6 (p < 0.0001), IL-8 (p < 0.05), and tumor necrosis factor (TNF)-α (p < 0.05) compared with the 'Uninjured' group., Conclusions: The results suggest that, following ARDS, there is an increase in the clonogenic capacity of MSCs to increase the available stem cell pool in vivo. However, MSCs harvested from subjects with ARDS seem to exhibit a diminished capacity to proliferate, express regenerative signals, and secrete pro/anti-inflammatory mediators.