The human mesenchymal stem cell (hMSC) secretome has pleiotropic effects which underpin their therapeutic potential. hMSC serum-free conditioned media (SFCM) has been determined to contain a variety of cytokines with roles in regeneration and suppression of inflammation. Physiological oxygen (physoxia) has been demonstrated to impact upon a number of facets of hMSC biology and we hypothesized that the secretome would be similarly modified. We tested a range of oxygen conditions; 21% O2 (air oxygen (AO)), 2% O2 (intermittent hypoxia (IH)) and 2% O2 Workstation (physoxia (P)) to evaluate their effect on hMSC secretome profiles. Total protein content of secretome was upregulated in IH and P (>3 fold vs AO) and IH (>1 fold vs P). Focused cytokine profiling indicated global upregulation in IH of all 31 biomolecules tested in comparison to AO and P with basic-nerve growth factor (bNGF) and granulocyte colony-stimulating factor (GCSF) (>3 fold vs AO) and bNGF and Rantes (>3 fold vs P) of note. Similarly, upregulation of interferon gamma-induced protein 10 (IP10) was noted in P (>3 fold vs AO). Interleukin-2 (IL2) and Rantes (in AO and P) and adiponectin, IL17a, and epidermal growth factor (EGF) (in AO only) were entirely absent or below detection limits. Quantitative analysis validated the pattern of IH-induced upregulation in vascular endothelial growth factor (VEGF), placental growth factor-1 (PIGF1), Tumor necrosis factor alpha (TNFa), IL2, IL4, and IL10 when compared to AO and P. In summary, modulation of environmental oxygen alters both secretome concentration and composition. This consideration will likely impact on delivering improved mechanistic understanding and potency effects of hMSC-based therapeutics.

The Human Mesenchymal Stem Cell (hMSC) secretome has pleiotropic effects underpinning its therapeutic potential. hMSC serum-free conditioned media (SFCM) contains a variety of cytokines, with previous studies linking a changed secretome composition to physoxia. The Jurkat T cell model allowed the efficacy of SFCM vs. serum-free media (SFM) in the suppression of immunological aspects, including proliferation and polarisation, to be explored. Cell growth in SFM was higher [(21% O2 = 5.3 × 105 ± 1.8 × 104 cells/mL) and (2% O2 = 5.1 × 105 ± 3.0 × 104 cells/mL)], compared to SFCM [(21% O2 = 2.4 × 105 ± 2.5 × 104 cells/mL) and (2% O2 = 2.2 × 105 ± 5.8 × 103 cells/mL)]. SFM supported IL-2 release following activation [(21% O2 = 5305 ± 211 pg/mL) and (2% O2 = 5347 ± 327 pg/mL)] whereas SFCM suppressed IL-2 secretion [(21% O2 = 2461 ± 178 pg/mL) and (2% O2 = 1625 ± 159 pg/mL)]. Anti-inflammatory cytokines, namely IL-4, IL-10, and IL-13, which we previously confirmed as components of hMSC SFCM, were tested. IL-10 neutralisation in SFCM restored proliferation in both oxygen environments (SFM/SFCM+antiIL−10 ~1-fold increase). Conversely, IL-4/IL-13 neutralisation showed no proliferation restoration [(SFM/SFM+antiIL−4 ~2-fold decrease), and (SFM/SFCM+antiIL−13 ~2-fold decrease)]. Present findings indicate IL-10 played an immunosuppressive role by reducing IL-2 secretion. Identification of immunosuppressive components of the hMSC secretome and a mechanistic understanding of their action allow for the advancement and refinement of potential future cell-free therapies.

Background Chronic disease states can share inflammation as an underlying pathology. Macrophages are associated with chronic inflammation; in general, M1 phenotype macrophage activity inhibits cell proliferation and causes tissue damage whilst M2 macrophages promote proliferation and tissue repair. Human mesenchymal stem/stromal cells (hMSCs) are of particular interest for immunoregulatory diseases, through their ability to suppress T cell proliferation. Methods The application of a physiological oxygen environment to hMSC cultures modulates their gene expression and results in an altered secretome. Consequently, physiological, 2% O2 hMSC SFCM was assessed in comparison to air oxygen (21% O2) produced SFCM and applied to activated THP-1 cells in 10% and 21% O2 to assess changes in macrophage differentiation. Results SFCM significantly suppressed proliferation and metabolic activity whilst promoting cell adherence compared to serum-free media (SFM) controls (p < 0.001). Terminal differentiation phenotype varied in an oxygen-dependent manner, with 21% air oxygen SFCM leading to an increased M1:M2 macrophage differentiation ratio, with cells displaying distinct pancake-like morphology, increased TNFa secretion (21% O2 = 922 ± 5 pg/mL, and 10% O2 = 373 ± 2 pg/mL) and CD197 expression (21% = 69.1%±6.4, and 10% = 18.6 ± 0.4%). In contrast, physiological oxygen SFCM led to the production of a greater M2:M1 macrophage differentiation ratio; displaying an elongated spindle shape, increased IL-10 secretion (21% O2 = 3780 ± 560 pg/mL, and 10% O2 = 9115 ± 889), and CD3 and CD14 expression. SFCM collected under 2% O2 suppressed pro-inflammatory transcriptional profiles through downregulation of TNFa, IL-1B, and IL-12B compared to 21% O2 collected SFCM. Conclusion Collectively, SFCM induced terminal macrophage differentiation in an oxygen-dependent manner. This effect was not completely replicated by the application of isolated candidate anti-inflammatory cytokines (IL-4, IL-10, IL-13 or TGFb) or a combinational cytomix, highlighting the complex effects of conditioned media products and further work needed to determine mechanism of action.