title: 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.
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Ghent University, Merelbeke, Belgium. catharina.deschauwer@ugent.be
authors.forename: Catharina
authors.initials: C
authors.lastname: De Schauwer
authors.forename: Evelyne
authors.initials: E
authors.lastname: Meyer
authors.forename: Pieter
authors.initials: P
authors.lastname: Cornillie
authors.forename: Sarne
authors.initials: S
authors.lastname: De Vliegher
authors.forename: Gerlinde R
authors.initials: GR
authors.lastname: van de Walle
authors.forename: Maarten
authors.initials: M
authors.lastname: Hoogewijs
authors.forename: Heidi
authors.initials: H
authors.lastname: Declercq
authors.forename: Jan
authors.initials: J
authors.lastname: Govaere
authors.forename: Kristel
authors.initials: K
authors.lastname: Demeyere
authors.forename: Maria
authors.initials: M
authors.lastname: Cornelissen
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D017667:Adipocytes / Q000166:cytology; D000818:Animals; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D002498:Centrifugation; D019902:Chondrocytes / Q000166:cytology; D003114:Colony-Forming Units Assay; D005312:Fetal Blood / Q000166:cytology*; D006736:Horses; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008954:Models, Biological; D010011:Osteocytes / Q000166:cytology