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

Abstract

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.

Cytokines in equine platelet lysate and related blood products

“…The decisive factors for clinical efficacy are presumably the growth factors contained in the PL, as well as anti-inflammatory cytokines, which can locally influence the inflammatory process ( 40 ). On the other hand, PL is already successfully used as a medium additive for cell culture of MSC, which can promote the cellular potency ( 6 , 19 , 41 , 42 ). While this has been largely attributed to the growth factors contained in the PL, e.g., VEGF ( 19 ), other mediators should be considered as well.…”

“…On the other hand, PL is already successfully used as a medium additive for cell culture of MSC, which can promote the cellular potency ( 6 , 19 , 41 , 42 ). While this has been largely attributed to the growth factors contained in the PL, e.g., VEGF ( 19 ), other mediators should be considered as well. As already described by Barrachina et al ( 43 ), stimulation with IFN-y or TNF-α in culture improves the immunomodulatory potency of MSC.…”

“…Overall, PL supports the unfolding of diverse biological activities of MSC so that they can exert proregenerative, anti-inflammatory, antifibrotic, and immunomodulatory effects ( 10 ). Accordingly, we have previously demonstrated that equine PL, produced by a scalable buffy-coat method, supports equine MSC proliferation and increases their pro-angiogenic potency ( 19 ). However, it has not been fully elucidated which PL components contribute to these beneficial effects.…”

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

“…Platelet lysate (PL) is a promising alternative to fetal bovine serum (FBS) as cell culture supplement for mesenchymal stromal cells (MSC). In previous studies, we were able to demonstrate that equine PL, produced by using the buffy coat method, promotes equine MSC proliferation, as well as their genetic stability and pro-angiogenic potential (1,2). MSC characteristics such as differentiation capacity and expression of surface markers were similar after cultivating the cells with PL or FBS (1).…”

“…In previous studies, we were able to demonstrate that equine PL, produced by using the buffy coat method, promotes equine MSC proliferation, as well as their genetic stability and pro-angiogenic potential (1,2). MSC characteristics such as differentiation capacity and expression of surface markers were similar after cultivating the cells with PL or FBS (1). PL contains a diversity of nutrients and growth factors such as TGF-β and PDGF (1,3).…”