Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

作     者：Elizabeth R A Glynn Alfredo Sanchez Londono Steven A Zinn Thomas A Hoagl Kristen E Govoni 

作者机构：Department of Animal ScienceUniversity of Connecticut3636 Horsebarn Road Ext.Unit 4040StorrsCT 06269-4040USA Department of Environmental and Population HealthTufts University North GraftonMA 01536USA 

出 版 物：《Journal of Animal Science and Biotechnology》 (畜牧与生物技术杂志（英文版）)

年 卷 期：2014年第5卷第2期

页      面：163-172页

核心收录：

学科分类：0710[理学-生物学] 0831[工学-生物医学工程（可授工学、理学、医学学位）] 0832[工学-食品科学与工程（可授工学、农学学位）] 1004[医学-公共卫生与预防医学(可授医学、理学学位)] 0905[农学-畜牧学] 09[农学] 0906[农学-兽医学] 0703[理学-化学] 0836[工学-生物工程] 

基　　金：supported by Storrs Agricultural Experiment Station Hatch Project CONS00844(KEG) 

主　　题：Bone marrow mesenchymal stem cells Cell culture Equine Osteoblasts Transcription factors 

摘      要：Background： The use of equine bone marrow mesenchymal stem cells （BMSC） is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine： 1） if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2） the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum （FBS） or autologous horse serum （HS）, and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 （BMP2）, dexamethasone （DEX）, or combination of the two. Alkaline phosphatase （ALP） activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-reloted tronscrJption foctor2 （Runx2）, osterix （Osx）, and T-box3 （Tbx3）. Data were analyzed by ANOVA. Results： Relative to control, FBS and HS increased cell number （133 ± 5 and 116 ± 5%, respectively; P 〈 0.001） and 5-bromo- 2＇-deoxyuridine （BrdU） incorporation （167 ± 6 and 120 ± 6%, respectively; P 〈 0.001）. Treatment with DEX increased ALP activity compared with control （1,638 ± 38%; P 〈 0.001）. In the absence and presence of Dex, BMP-2 did not alter ALP activity （P 〉 0.8）. Runt-reloted transcription foctor2 expression increased 3-fold （P 〈 0.001） by d 6 of culture. Osterix expression increased 94old （P 〈 0.05） by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 （P 〈 0.01）; however expression was reduced 4-fold at d 18 （P 〈 0.01）. Conclusions： Dexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.