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 E₂ (PGE₂), substance P (SP), their combination and IFNγ. PGE₂/SP was the best combination to keep or enhance the mesodermal lineage differentiation of eAMSC. Alongside with this, preconditioning of eMSC with PGE₂ 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 naïve and preconditioned eAMSC expressed genes related with immune properties, such as MHC-I, PTGES, IL6, IL1A, TNFα and IL8 assessed by qPCR. Only TNFα 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 naïve eAMSC. In the experiments of this research, we investigated the secreted proteomic profile of naïve 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 naïve eAMSC as well as among treatments. In overall, compared to naïve cells, the protein profile of preconditioned cells resembled the mesenchymal-epithelial transition (MET). Here we showed that the combined use of PGE₂ 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”.

间充质干细胞（MSC）由于具有免疫调节特性而在人类和动物的再生疗法中得到了广泛应用，它们在促炎性环境中被激活。已经设计了不同的预处理策略来增强MSC的免疫调节特性。在这项研究中，我们评估了用前列腺素E ₂（PGE ₂），P物质（SP），它们的组合和IFNγ进行体外预​​处理之前对马脂肪MSC（eAMSC）的免疫学特性。PGE ₂ / SP是保持或增强eAMSC的中胚层谱系分化的最佳组合。除此之外，用PGE ₂和SP预处理eMSC不会影响干细胞MSC表面表型的表达：CD90⁺，CD44 ⁺，MHC I类⁺，MHC II类^-和CD45 ^-，通过细胞计数评估。幼稚和预处理过的eAMSC均表达与免疫特性相关的基因，例如通过qPCR评估的MHC-1，PTGES，IL6，IL1A，TNFα和IL8。在处理过的细胞中只有TNFα表达不足，而其他标记则过表达或未改变。在任何情况下均未检测到MHC-II表达。暴露于活化的外周血单个核细胞（PBMC）的预处理eAMSC的抗增殖作用表明SP治疗显着抑制LPS刺激的PBMC的增殖。当用Poly I：C刺激eAMSC时，所有处理均显着抑制了刺激的PBMC的增殖（p <0.05）。预处理过的eAMSC和PBMC之间的直接接触（共培养），导致明显更多的移位（CD4 / CD25 / FOXP3）⁺T调节性PBMC比单纯的eAMSC更重要。在这项研究的实验中，我们调查了幼稚和预处理过的eAMSC的分泌蛋白质组学特征，在蛋白质组学分析中发现了42种上调和40种下调的蛋白质。我们的蛋白质组学数据显示，与单纯的eAMSC以及不同处理相比，暴露于不同处理的MSC分泌模式发生了深远的变化。总体而言，与原始细胞相比，预处理细胞的蛋白质谱类似于间充质-上皮转化（MET）。在这里，我们显示PGE ₂和SP的组合使用在总体上引起了抗炎标记物的最高表达，并导致预处理eAMSC中T调节表型的获取增加，而不影响其“干性”。