Priming of MSC with inflammatory and damage related factors: a model to study changes of gene and protein patterns

Mesenchymal stromal cells (MSCs) are a major cell source for regenerative medicine in various therapeutic approaches. Recent studies show that priming of MSCs can improve their regenerative potential. We primed MSCs with a mix of inflammatory-associated molecules (IC) and a mix of damage-associated proteins (DC) to investigate how each priming affects MSC"s transcriptome and protein expression. Moreover, we tried to identify which single factor of the IC is the main mediator of the IC effects.

MSCs were incubated with IC, consisting of IL-1β, TNF-α, IL-6, CXCL8, C3a, C5a and DC, consisting of HMGB1 and histones for 6 hours and 24 hours. Differently expressed gene analysis of these datasets in comparison with an unstimulated control for each incubation period were performed. Proteins of 20 selected immunomodulatory genes upregulated (fold change ≥ 3, false discovery rate ≤ 0.01) by IC were analysed by measurement of supernatant concentrations with magnetic-bead-based multiplex assays. Pathway analysis of differently expressed genes was conducted. Main mediators of the IC were identified in silico and via incubating MSCs with the individual IC components and measurement of certain supernatant protein concentrations.

IC and DC priming resulted in significant transcriptomic changes: After 6 and 24 hours 447 genes were upregulated by IC, 77 by DC and 44 by IC and DC. DC and IC induced different patterns with stronger effects by IC in terms of number of affected genes and the magnitude of fold-change. In contrast to DC, IC increased concentration of 13 of 20 selected proteins (CCL7, CCL20, CXCL1, CXCL5, CXCL6, CXCL8, MMP3, IL-6, IL-27, CSF2, CSF3, LIF). Upstream regulator analysis revealed IL-1β and TNF-α as main mediators of IC effects. The canonical pathways affected by IC with highest Z-scores (QIAGEN IPA core analysis) were IL-17A signaling in fibroblasts, IL-17 signaling and IL-6 signaling. DC only affected TREM1-Signaling after both 6 and 24 hours.

Priming of MSCs with inflammatory related molecules affects the transcriptome and protein expression. All upregulated proteins analysed in the supernatant of IC primed MSCs are relevant for immune response and have autocrine and paracrine effects. Therefore, we conclude that priming of MSCs with IC might improve their regenerative potential in inflammatory diseases. Future research should focus on reduction of IC components to ideally a single priming agent to optimize MSCs for therapy.

The authors declare no commercial, proprietary or financial interest.

SE, MW, MK, MR and HS work for the University Hospital Ulm. AG works for the Ulm University. This work is supported by Deutsche Forschungsgemeinschaft grant CRC1149 "Danger Response, Disturbance Factors and Regenerative Potential after Acute Trauma", project number 251293561. The materials presented and views expressed here are the responsibility of the authors only.