title: Population doubling level-dependent change of secreted glycosaminoglycan in equine bone marrow-derived mesenchymal stem cells.
abstract: In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amount of chondroitin sulfate. Increase of PDL downregulated the expression of MSC CD markers, including CD44, CD73, CD90, CD105, and CD146, along with loss of differentiation capacity. Thus, the effect of hyaluronan supplement to the growth medium on both expression of CD markers and the tri-lineage potential of BMSCs was evaluated. Expression of CD73 and CD90 was preserved by continuous addition of hyaluronan to the growth medium, whereas mRNA levels corresponding to CD44, CD105 and CD146 were not preserved by supplementation of hyaluronan. BMSCs subcultured with hyaluronan-supplemented growth medium to PDL-12 showed osteogenic capacity, however adipogenic and chondrogenic activities at PDL-12 were not preserved by exogenous hyaluronan. These results suggest that downregulation of CD44, CD105 and CD146 might not affect the osteogenic capacity. Taken together, the results suggested that supplementation of hyaluronan to the growth medium might be effective at maintaining the osteogenic capacity of equine BMSCs.
authors.affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sasao
authors.affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: Sayako
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: Shihori
authors.initials: S
authors.lastname: Miyabara
authors.affiliation: Laboratory of Clinical Science and Pathobiology, Equine Research Institute, Japan Racing Association, Tochigi 320-8056, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kasashima
authors.affiliation: Laboratory of Clinical Science and Pathobiology, Equine Research Institute, Japan Racing Association, Tochigi 320-8056, Japan.
authors.forename: Atsutoshi
authors.initials: A
authors.lastname: Kuwano
authors.affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Arai
keywords: CD; hyaluronan; osteogenesis; stemness