Impact of platelet lysate on immunoregulatory characteristics of equine mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSC) play an increasing role in the treatment of immune-mediated diseases and inflammatory processes. They regulate immune cells via cell-cell contacts and by secreting various anti-inflammatory molecules but are in turn influenced by many factors such as cytokines. For MSC culture, platelet lysate (PL), which contains a variety of cytokines, is a promising alternative to fetal bovine serum (FBS). We aimed to analyze if PL with its cytokines improves MSC immunoregulatory characteristics, with the perspective that PL could be useful for priming the MSC prior to therapeutic application. MSC, activated peripheral blood mononuclear cells (PBMC) and indirect co-cultures of both were cultivated in media supplemented with either PL, FBS, FBS+INF-γ or FBS+IL-10. After incubation, cytokine concentrations were measured in supernatants and control media. MSC were analyzed regarding their expression of immunoregulatory genes and PBMC regarding their proliferation and percentage of FoxP3+ cells. Cytokines, particularly IFN-γ and IL-10, remained at high levels in PL control medium without cells but decreased in cytokine-supplemented control FBS media without cells during incubation. PBMC released IFN-γ and IL-10 in various culture conditions. MSC alone only released IFN-γ and overall, cytokine levels in media were lowest when MSC were cultured alone. Stimulation of MSC either by PBMC or by PL resulted in an altered expression of immunoregulatory genes. In co-culture with PBMC, the MSC gene expression of COX2, TNFAIP6, IDO1, CXCR4 and MHC2 was upregulated and VCAM1 was downregulated. In the presence of PL, COX2, TNFAIP6, VCAM1, CXCR4 and HIF1A were upregulated. Functionally, while no consistent changes were found regarding the percentage of FoxP3+ cells, MSC decreased PBMC proliferation in all media, with the strongest effect in FBS media supplemented with IL-10 or IFN-γ. This study provides further evidence that PL supports MSC functionality, including their immunoregulatory mechanisms. The results justify to investigate functional effects of MSC cultured in PL-supplemented medium on different types of immune cells in more detail.

Cytokines in equine platelet lysate and related blood products.

In equine medicine, the use of regenerative therapeutics has gained growing attention, but is still a new and complex field with room for improvement. Platelet lysate (PL) can be used as therapeutic agent but is also a promising supplement for the culture of multipotent mesenchymal stromal cells. To enable a targeted use of PL both in clinic and laboratory, it is crucial to learn more details on its effective ingredients. While so far, mainly growth factor components have been analyzed in platelet-based products such as PL, the current study focuses on the content of cytokines in serum, plasma, platelet concentrate and PL. Blood was harvested from 20 clinically healthy horses and subjected to blood count and chemistry analysis, as well as to further processing to PL. Plasma and platelet concentrate were produced by a buffy-coat-based method and PL was produced from the concentrate by freeze-thawing. Samples from each horse were analyzed regarding interleukin (IL)-1ß, -4, -6 and -10, interferon-γ and tumor necrosis factor-α concentrations using sandwich ELISAs. Cytokine concentrations in serum, plasma, concentrate and PL were similar and correlated significantly. However, there was a large inter-individual variability in cytokine concentrations between the different donor horses. The samples from some donor animals had overall very high cytokine concentrations, while samples from other donors had no measurable cytokine ingredient. This pattern was observed for all cytokines. There was a noticeable link between high cytokine concentrations in the blood products and abnormal findings in blood chemistry. Cytokine concentrations in samples from horses with abnormal findings were significantly higher than in samples from the remaining horses. The interindividual differences in cytokine concentrations could be highly relevant when using PL for therapy and cell culture, as the mode of action of the PL is likely changed depending on the presence of pro- and anti-inflammatory cytokines. Blood chemistry might be useful to predict cytokine concentrations in blood products.

Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate.

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same.

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-ß1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

Rho/ROCK Inhibition Promotes TGF-ß3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-ß, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-ß3-induced tenogenic differentiation. The MSC were incubated with and without TGF-ß3 (10 ng/ml), Y-27632 (10 µM), or both. On day 1 and day 3, the signalling pathway of TGF-ß and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-ß3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-ß3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-ß3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-ß3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.