Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/μL) or basal PL (BPL; 177 × 103 platelets/μL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P < 0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (P < 0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.

中文翻译：

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同种异体血小板裂解物对马骨髓间充质干细胞特征的影响，包括免疫原性和免疫调节基因表达谱。

间充质干细胞（MSCs）的离体繁殖需要补充培养基。胎牛血清（FBS）长期以来一直是金标准补充剂，但由于伦理和安全问题，其使用受到质疑。已经提出使用血小板裂解物（PL）替代FBS，但是关于其对马MSC特性包括其免疫特性的影响知之甚少。这项工作的目的是第一次研究同种异体PL对马骨髓来源的MSC（eBM-MSC）的免疫原性和免疫调节基因表达谱的影响，以及它们的增殖能力，表型标记和生存能力。 -低温保存。在eBM-MSC（n = 3）培养物中补充20％的同种异体合并浓缩PL（CPL; 591×103血小板/μL）或基础PL（BPL; 使用10％FBS补充作为三份供体的177×103血小板/μL）。通过计算直至第3代（P3）的细胞倍增时间（DT）和通过3-（4,5-二甲基噻唑-2-基）-2,5-评估eBM-MSC在三种条件下的增殖能力。在P3处进行溴化二苯基四唑鎓（MTT）分析。在第15、30和60天评估用CPL或FBS冷冻保存后的eBM-MSC的生存力。通过RT-qPCR在P3中评估eBM-MSC的基因表达谱，以表征，免疫原性和免疫调节标记。在MTT分析中，CPL培养的细胞具有比FBS或BPL显着更高的增殖能力（P <0.001）。在所有时间点，在FBS和CPL中培养和保存的细胞之间，低温保存后的存活率相似。在这三个条件下，MSC表征标记的基因表达相似。与FBS和BPL相比，在CPL条件下，免疫原性标记MHC-1，MHC-II和CD40的基因表达略有增加（无显着性）。CPL条件显示编码免疫调节分子VCAM-1（无显着性）和IL-6（P <0.05）的基因表达较高，与COX-2相似。而iNOS和IDO在任何情况下都不表达。总之，用同种异体CPL替代FBS作为eBM-MSC体外繁殖的补充剂，可提供适当的增殖和冷冻保存，并适度上调免疫调节标记的基因表达，从而构成使用FBS的潜在合适替代品。