Optimization of the isolation, culture, and characterization of equine umbilical cord blood mesenchymal stromal cells.
Abstract: Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.
Publication Date: 2011-08-26 PubMed ID: 21870941DOI: 10.1089/ten.tec.2011.0052Google Scholar: Lookup
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- Journal Article
Summary
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This research primarily focuses on optimizing the process of isolating, growing, and examining mesenchymal stromal cells (also known as MSCs) from the blood in a horse’s umbilical cord. The study compares different methods of isolation and culture conditions to conclude the most effective method.
Overview of Studies on Mesenchymal Stromal Cells
- Mesenchymal stromal cells (MSCs) are seen as a major breakthrough for their potential applications in cellular therapy. Their ability to differentiate into different types of cells like bones, cartilage, and fat cells gives them the potential to support new clinical concepts.
- While there have been numerous studies and procedures developed to isolate these cells from human umbilical cord blood, limited data is available about the same process in horses.
Isolation Methods Compared
- In this study, four isolation methods were compared: the sedimentation method using hydroxyethyl starch, a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl), and two density gradient separation methods (Ficoll-Paque and Percoll).
- With all four methods, researchers were able to establish adherent cell colonies. However, the use of Percoll separation resulted in significantly more MSC colonies.
Culture Conditions
- Researchers also studied the impact of culture conditions on MSC recovery, including factors such as cell density and medium or serum coating of the wells. None of these factors significantly affected the recovery of MSCs.
Phenotyping MSCs
- The MSCs isolated using Percoll were then made to differentiate into osteogenic (bone-forming), chondrogenic (cartilage-forming), and adipogenic (fat-forming) lineages to examine their versatility.
- The characteristics of these MSCs were determined by analyzing their expression of different cell protein markers. The MSCs were positive for CD29, CD44, and CD90 markers while they did not express CD79α, Macrophage/Monocyte, and MHC II markers.
Conclusion
- The research study concludes by providing optimized protocols for isolating, culturing, and characterizing MSCs from horse umbilical cord blood. This forms a base for further research in the use of these cells in equine medicine.
Cite This Article
APA
De Schauwer C, Meyer E, Cornillie P, De Vliegher S, van de Walle GR, Hoogewijs M, Declercq H, Govaere J, Demeyere K, Cornelissen M, Van Soom A.
(2011).
Optimization of the isolation, culture, and characterization of equine umbilical cord blood mesenchymal stromal cells.
Tissue Eng Part C Methods, 17(11), 1061-1070.
https://doi.org/10.1089/ten.tec.2011.0052 Publication
Researcher Affiliations
- Department of Reproduction, Obstetrics and Herd Health, Ghent University, Merelbeke, Belgium. catharina.deschauwer@ugent.be
MeSH Terms
- Adipocytes / cytology
- Animals
- Cell Adhesion
- Cell Count
- Cell Culture Techniques / methods
- Cell Differentiation
- Cell Proliferation
- Cell Separation / methods
- Cell Shape
- Cell Survival
- Cells, Cultured
- Centrifugation
- Chondrocytes / cytology
- Colony-Forming Units Assay
- Fetal Blood / cytology
- Horses
- Immunophenotyping
- Mesenchymal Stem Cells / cytology
- Models, Biological
- Osteocytes / cytology
Citations
This article has been cited 14 times.- Heyman E, Meeremans M, Devriendt B, Olenic M, Chiers K, De Schauwer C. Validation of a color deconvolution method to quantify MSC tri-lineage differentiation across species. Front Vet Sci 2022;9:987045.
- Taylor SD, Serpa PBS, Santos AP, Hart KA, Vaughn SA, Moore GE, Mukhopadhyay A, Page AE. Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses. J Vet Intern Med 2022 Jul;36(4):1491-1501.
- 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.
- Meeremans M, Van Damme L, De Spiegelaere W, Van Vlierberghe S, De Schauwer C. Equine Tenocyte Seeding on Gelatin Hydrogels Improves Elongated Morphology. Polymers (Basel) 2021 Feb 28;13(5).
- Manda JK, Tchokonte-Nana V. Immunohistochemical characterization of pancreatic duodenal homeobox protein-1, neurogenin-3, and insulin protein expressions in islet-mesenchymal cell in vitro: a morphochronological evaluation. Iran J Basic Med Sci 2018 Nov;21(11):1126-1132.
- Barboni B, Russo V, Berardinelli P, Mauro A, Valbonetti L, Sanyal H, Canciello A, Greco L, Muttini A, Gatta V, Stuppia L, Mattioli M. Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance. Cell Transplant 2018 Jan;27(1):93-116.
- Tessier L, Bienzle D, Williams LB, Koch TG. Phenotypic and immunomodulatory properties of equine cord blood-derived mesenchymal stromal cells. PLoS One 2015;10(4):e0122954.
- Mohanty N, Gulati BR, Kumar R, Gera S, Kumar S, Kumar P, Yadav PS. Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood. Cytotechnology 2016 Aug;68(4):795-807.
- De Schauwer C, Goossens K, Piepers S, Hoogewijs MK, Govaere JL, Smits K, Meyer E, Van Soom A, Van de Walle GR. Characterization and profiling of immunomodulatory genes of equine mesenchymal stromal cells from non-invasive sources. Stem Cell Res Ther 2014 Jan 13;5(1):6.
- Mohanty N, Gulati BR, Kumar R, Gera S, Kumar P, Somasundaram RK, Kumar S. Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood. In Vitro Cell Dev Biol Anim 2014 Jun;50(6):538-48.
- Spaas JH, Guest DJ, Van de Walle GR. Tendon regeneration in human and equine athletes: Ubi Sumus-Quo Vadimus (where are we and where are we going to)?. Sports Med 2012 Oct 1;42(10):871-90.
- Marmotti A, Mattia S, Bruzzone M, Buttiglieri S, Risso A, Bonasia DE, Blonna D, Castoldi F, Rossi R, Zanini C, Ercole E, Defabiani E, Tarella C, Peretti GM. Minced umbilical cord fragments as a source of cells for orthopaedic tissue engineering: an in vitro study. Stem Cells Int 2012;2012:326813.
- Heyman E, Olenic M, De Vlieghere E, De Smet S, Devriendt B, Thorrez L, De Schauwer C. Donor age and breed determine mesenchymal stromal cell characteristics. Stem Cell Res Ther 2025 Feb 28;16(1):99.
- Heyman E, Devriendt B, De Vlieghere E, Goethals K, Van Poucke M, Peelman L, De Schauwer C. Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation. NPJ Sci Food 2024 Oct 1;8(1):70.
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