Sasao Takafumi, Fukuda Yuki, Yoshida Sayako, Miyabara Shihori, Kasashima Yoshinori, Kuwano Atsutoshi, Arai Katsuhiko
Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
Laboratory of Clinical Science and Pathobiology, Equine Research Institute, Japan Racing Association, Tochigi 320-8056, Japan.
J Equine Sci. 2015;26(3):73-80. doi: 10.1294/jes.26.73. Epub 2015 Sep 30.
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.
在使用间充质干细胞（MSCs）移植的再生医学中，调节MSCs质量的重要性已得到充分认识；然而，在马医学中，关于群体倍增水平（PDL）与MSCs干性之间的关系却知之甚少。在本研究中，我们发现骨髓来源的间充质干细胞（BMSCs）分泌的糖胺聚糖（GAG）量随PDL的增加而减少。酶消化和二维电泳显示，BMSCs产生的GAG的主要成分是透明质酸，还有少量硫酸软骨素。PDL的增加下调了包括CD44、CD73、CD90、CD105和CD146在内的MSC CD标志物的表达，同时分化能力丧失。因此，评估了在生长培养基中添加透明质酸对BMSCs的CD标志物表达和三系分化潜能的影响。通过在生长培养基中持续添加透明质酸可保留CD73和CD90的表达，而添加透明质酸并不能保留与CD44、CD105和CD146相对应的mRNA水平。用添加透明质酸的生长培养基传代培养至PDL-12的BMSCs显示出成骨能力，然而，外源性透明质酸并不能保留PDL-12时的成脂和成软骨活性。这些结果表明，CD44、CD105和CD146的下调可能不会影响成骨能力。综上所述，结果表明在生长培养基中添加透明质酸可能对维持马BMSCs的成骨能力有效。
