Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells

Naskou et al. Stem Cell Research & Therapy (2018) 9:75 https://doi.org/10.1186/s13287-018-0823-3 RESEARCH Open Access Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells Maria C. Naskou1, Scarlett M. Sumner1, Anna Chocallo1, Hannah Kemelmakher1, Merrilee Thoresen1, Ian Copland2ˆ, Jacques Galipeau3 and John F. Peroni1* Abstract Background: Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Methods: Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Results: Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. Conclusion: ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS. Keywords: Equine platelet apheresis, Equine platelet lysate, Mesenchymal stem cells, Fetal bovine serum, Cell culture * Correspondence: ˆDeceased 1 Department of Large Animal Medicine, Veterinary Medical Center, College of Veterinary Medicine, University of Georgia, 2200 College Station Road, Athens, GA 30602, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Naskou et al. Stem Cell Research & Therapy (2018) 9:75 Background Mesenchymal stem cells (MSCs) are multipotent selfrenewing cells that have been implicated in orchestrating the repair of damaged tissues by modulating the endogenous repair process through interacting with the inflammatory response of the injured tissue [1–3]. The preparation of MSCs before clinical application requires their primary isolation and ex vivo expansion to propagate an adequate number of cells for transplantation. Fetal bovine serum (FBS) is the current gold standard culture additive used as a source of growth factors, hormones, and vital nutrients to support MSC expansion in the laboratory [4–9]. Unfortunately, there is concerning evidence to show that FBS contains endotoxins (such as lipopolysaccharide (LPS)) and xenogeneic antigens that may alter the phenotype of MSCs grown in FBS, rendering these cells immunogenic [10–12]. This may prompt the immune system to reject MSCs following introduction into the recipient, even when the delivered MSCs are autologous to the host. The transplantation of MSCs cultured with traditional culture techniques is also a potential route of transmission of FBS-derived animal pathogens, such as prions and viruses [13–15]. Furthermore, the Food and Drug Administration (FDA) has encouraged the use of xenoprotein-free culture conditions for the expansion of MSCs in humans to avoid adverse effects related to FBS [16]. These facts, together with the rising cost of FBS and ethical concerns related to the manufacturing of FBS, underpin the rationale behind the development of FBS-free media to support the expansion of MSCs for clinical purposes. To this end, several studies have investigated the use of platelet-derived products, such as platelet lysate (PL), obtained following the lysis of platelets from platelet concentrates or platelet-rich plasma (PRP) as a media supplement for the in vitro culture of various types of cells [4]. Human PL is a reportedly superior alternative to FBS and serum for the ex vivo expansion of MSCs which, in the presence of PL, maintain their differentiation potential, immune-phenotype, and immunomodulatory activities [9, 17–19]. In addition to the major role platelets play in hemostasis, they are a principal source of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), attachment factors, and enzymes found in serum. These factors can enhance the recruitment, proliferation, and differentiation of MSCs, but also exhibit anti-inflammatory and angiogenic properties [20–22]. In veterinary medicine, equine PL (ePL) obtained from whole blood via two-step centrifugation can be used instead of FBS for the culture of equine bone marrowderived MSCs and the short-term expansion of equine cord blood MSCs [6, 23]. Differences in the preparation Page 2 of 13 process of PL such as platelet separation methods (apheresis versus two-step centrifugation), platelet activation (freeze/thaw cycles versus calcium chloride), and removal of platelet fragments, can affect PL growth factor concentrations and therefore influence the proliferative rate and the differentiation capacity of MSCs [5, 24, 25]. We have recently shown that (...truncated)