The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells

Abstract

Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.

Effects of mesenchymal stem cell therapy on alopecia areata in cellular and hair follicle organ culture models

“…Phosphorylation of IL‐15 was reduced by hHMSCs alone but enhanced by hHMSCs under the IFN‐γ–prone environment in this study. This result is consistent with previous reports in that the IFN‐γ microenvironment stimulates MSCs to produce more pro‐inflammatory cytokines …”

“…Interestingly, phosphorylation of cyclin D in response to hHMSC was shown to be significantly higher in IFN‐γ–pretreated hDPCs compared to IFN‐γ–untreated hDPCs. This is consistent with previous studies demonstrating that secretion ability of MSCs is enhanced by certain cellular environments such as hypoxia or with IFN‐γ treatment . Although we did not examine the effect of MSCT on the ERK and MAPK pathways, MSCT might activate these pathways, which contributes to cell cycle activation through targeting such as cyclin D1.…”

Time Course of Changes in Sorafenib‐Treated Hepatocellular Carcinoma Cells Suggests Involvement of Phospho‐Regulated Signaling in Ferroptosis Induction

“…Phosphorylated peptides were eluted from the tip‐based column using 5% then 10% ammonium hydroxide in 20% ACN. Following enrichment, samples were again desalted using Nestgroup C18 Macrospin columns (Southborough, MA) as previously described [ 29 ] based on manufacturer protocol and lyophilized to dryness before peptide quantitation via NanoDrop spectrophotometry (ThermoFisher Scientific) and LC‐MS/MS acquisition.…”

“…Next, samples were incubated for 30 min at 60 °C followed by a methanol chloroform protein extraction as previously described. [ 29 ] Briefly, methanol and chloroform were added to the lysate in a 4:4:1 ratio of lysate:methanol:chloroform. Samples were centrifuged for 1 min at 16 000 × g and two additional methanol washes were performed to remove lipids and other potentially contaminating compounds prior to downstream processing.…”

Water as a reactant in the differential expression of proteins in cancer

“…Moreover, secreted proteins in hypoxia are more often relatively oxidized (Table 2). Hypoxia is often associated with the downregulation of mitochondrial proteins (66,67). These have a relatively low Z C compared to other subcellular fractions such as cytoplasm and nucleus (26), so their downregulation would tend to produce overall more oxidized proteins.…”