Abstract

Mesenchymal stem cells (MSC) are modern tools in regenerative therapies of humans and animals owed to their immunomodulatory properties, which are activated in a pro-inflammatory environment. Different preconditioning strategies had been devised to enhance the immunomodulatory properties of MSC. In this research, we evaluated the immunological attributes of equine adipose MSC (eAMSC) before and after preconditioning in vitro with prostaglandin E-2 (PGE2), substance P (SP), their combination and IFN gamma. PGE(2)/SP was the best combination to keep or enhance the mesodermal lineage differentiation of eAMSC. Alongside with this, preconditioning of eMSC with PGE(2) and SP did not affect expression of stemness MSC surface phenotype: CD90(+), CD44(+), MHC class I+, MHC class II- and CD45(-), assessed by cytometry. Both naive and preconditioned eAMSC expressed genes related with immune properties, such as MHC-I, PTGES, IL6, IL1A, TNF alpha and IL8 assessed by qPCR. Only TNF alpha was under expressed in treated cells, while the other markers were either overexpressed or not changed. In no cases MHC-II expression was detected. The antiproliferative effect of preconditioned eAMSC exposed to activated peripheral blood mononuclear cells (PBMC) showed that SP treatment significantly inhibited proliferation of LPS stimulated PBMC. When eAMSC were stimulated with Poly I:C, all the treatments significantly inhibited proliferation of stimulated PBMC (p 0.05). Direct contact (coculture) between the preconditioned eAMSC and PBMC, induced a shift of significantly more (CD4/CD25/FOXP3)(+) T-regulatory PBMC than naive eAMSC. In the experiments of this research, we investigated the secreted proteomic profile of naive and preconditioned eAMSC, 42 up-regulated and 40 down-regulated proteins were found in the proteomic assay. Our proteomic data revealed profound changes in the secretory pattern of MSC exposed to different treatments, compared to naive eAMSC as well as among treatments. In overall, compared to naive cells, the protein profile of preconditioned cells resembled the mesenchymal-epithelial transition (MET). Here we showed that the combined use of PGE(2) and SP provoked in overall the highest expression of anti-inflammatory markers as well as lead to an increased acquisition of a T-regulatory phenotype in preconditioned eAMSC without affecting their "stemness".