Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.
Abstract: 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 × 10 platelets/μL) or basal PL (BPL; 177 × 10 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 (P) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P. 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 P 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.
Copyright © 2019 Elsevier B.V. All rights reserved.
Publication Date: 2019-09-21 PubMed ID: 31563725DOI: 10.1016/j.vetimm.2019.109944Google Scholar: Lookup
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Summary
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The research discusses the use of allogeneic platelet lysate (CPL) in place of fetal bovine serum (FBS) for the culture and preservation of equine mesenchymal stem cells (MSCs). This method showed favorable results in MSCs’ proliferation and cryopreservation, as well as a slight increase in the expression of certain immunomodulatory markers.
Platelet Lysate as Fetal Bovine Serum Substitute
- The study points out that ex vivo propagation of MSCs typically involves the use of a supplement, often fetal bovine serum (FBS).
- However, due to various ethical and safety constraints associated with FBS, researchers have been exploring alternatives, one of which is platelet lysate (PL).
- Platelet lysate, particularly allogeneic platelet lysate, was the main focus of this study as it was used in the cultivation of equine bone marrow-derived mesenchymal stem cells (eBM-MSCs).
Study Design and Methodology
- The research worked with three cultures of eBM-MSCs. These were supplemented with either 20% CPL, basal PL (BPL), or 10% FBS—the latter served as the control group.
- The researchers examined the proliferative ability of eBM-MSCs under these conditions via cell doubling times and the MTT assay, an test to assess the viability of cells.
- The cells’ post-cryopreservation viability with CPL or FBS was also studied over 15, 30, and 60-day periods.
- The researchers evaluated the gene expression profiles for characterization, immunogenic, and immunomodulatory markers.
Results and Conclusion
- The results showed that eBM-MSCs cultivated in CPL had a significantly higher proliferation rate compared to those in FBS or BPL.
- Post-cryopreservation viability of the cells cultured in CPL was akin to those preserved in FBS at all studied timepoints.
- The gene expression of MSC characterization markers remained similar across all test conditions, however, the CPL condition showed a non-significant increase in expression of some immunogenic markers, and established higher expression of genes coding for certain immunomodulatory markers.
- The research, therefore, concluded that replacing FBS with allogeneic CPL in eBM-MSCs could provide a viable alternative for cell proliferation and preservation, but further studies are needed to understand the effects of CPL supplementation on equine MSCs immunological profile.
Cite This Article
APA
Yaneselli K, Barrachina L, Remacha AR, Algorta A, Vitoria A, Cequier A, Romero A, Vázquez FJ, Maisonnave J, Rodellar C.
(2019).
Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.
Vet Immunol Immunopathol, 217, 109944.
https://doi.org/10.1016/j.vetimm.2019.109944 Publication
Researcher Affiliations
- Área Inmunología, Facultad de Veterinaria, Universidad de la República, 11600, Montevideo, Uruguay. Electronic address: kyaneselli@fvet.edu.uy.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain. Electronic address: lbarrach@unizar.es.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain. Electronic address: aremacha@unizar.es.
- Área Inmunología, Facultad de Veterinaria, Universidad de la República, 11600, Montevideo, Uruguay. Electronic address: agustinaalgorta@gmail.com.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain; Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain. Electronic address: avm@unizar.es.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain. Electronic address: alinacs@unizar.es.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain; Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain. Electronic address: aromerol@unizar.es.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain; Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain. Electronic address: pvazquez@unizar.es.
- Área Inmunología, Facultad de Veterinaria, Universidad de la República, 11600, Montevideo, Uruguay. Electronic address: jacmaiso@gmail.com.
- Laboratorio de Genética Bioquímica (LAGENBIO), Instituto Agroalimentario de Aragón IA2 - Instituto de Investigación Sanitaria de Aragón IIS, Universidad de Zaragoza, 50013, Zaragoza, Spain. Electronic address: rodellar@unizar.es.
MeSH Terms
- Animals
- Blood Platelets / chemistry
- Bone Marrow Cells / cytology
- Bone Marrow Cells / immunology
- Cell Culture Techniques
- Cell Differentiation
- Cell Extracts / chemistry
- Cell Proliferation
- Cell Survival
- Culture Media / chemistry
- Female
- Gene Expression Profiling
- Horses
- Male
- Mesenchymal Stem Cells / cytology
- Mesenchymal Stem Cells / immunology
- Transcriptome
Citations
This article has been cited 9 times.- Moellerberndt J, Hagen A, Niebert S, Büttner K, Burk J. Cytokines in equine platelet lysate and related blood products. Front Vet Sci 2023;10:1117829.
- Jammes M, Contentin R, Cassé F, Galéra P. Equine osteoarthritis: Strategies to enhance mesenchymal stromal cell-based acellular therapies. Front Vet Sci 2023;10:1115774.
- Even KM, Gaesser AM, Ciamillo SA, Linardi RL, Ortved KF. Comparing the immunomodulatory properties of equine BM-MSCs culture expanded in autologous platelet lysate, pooled platelet lysate, equine serum and fetal bovine serum supplemented culture media. Front Vet Sci 2022;9:958724.
- Hagen A, Niebert S, Brandt VP, Holland H, Melzer M, Wehrend A, Burk J. Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate. Front Vet Sci 2022;9:890302.
- Pilgrim CR, McCahill KA, Rops JG, Dufour JM, Russell KA, Koch TG. A Review of Fetal Bovine Serum in the Culture of Mesenchymal Stromal Cells and Potential Alternatives for Veterinary Medicine. Front Vet Sci 2022;9:859025.
- Hagen A, Holland H, Brandt VP, Doll CU, Häußler TC, Melzer M, Moellerberndt J, Lehmann H, Burk J. Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same. Animals (Basel) 2022 Jan 13;12(2).
- Hagen A, Lehmann H, Aurich S, Bauer N, Melzer M, Moellerberndt J, Patané V, Schnabel CL, Burk J. Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture. Front Bioeng Biotechnol 2020;8:613621.
- Yaneselli K, Ávila G, Rossi A, Rial A, Castro S, Estradé MJ, Suárez G, Algorta A. Impact of different formulations of platelet lysate on proliferative and immune profile of equine mesenchymal stromal cells. Front Vet Sci 2024;11:1410855.
- Duysens J, Graide H, Niesten A, Mouithys-Mickalad A, Deby-Dupont G, Franck T, Ceusters J, Serteyn D. Culture and Immunomodulation of Equine Muscle-Derived Mesenchymal Stromal Cells: A Comparative Study of Innovative 2D versus 3D Models Using Equine Platelet Lysate. Cells 2024 Jul 31;13(15).
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