Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells
Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells
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DOI：
10.1002/art.23370
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发表时间：
2008-05-01
影响因子：
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通讯作者：
Tuan, Rocky S.
中科院分区：
文献类型：
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作者：
Mueller, Michael B.;Tuan, Rocky S.
目标。间充质干细胞（MSCs）是软骨组织工程中很有前途的候选细胞。软骨肥大标志物（如X型胶原）在软骨形成过程中的表达引起了对MSCs组织工程应用的关注，因为肥大会导致细胞凋亡和骨化。为了分析骨髓间充质干细胞肥大的生物学基础，我们使用一系列肥大相关标记物研究了软骨化骨髓间充质干细胞对已知影响软骨细胞肥大的培养条件的反应。人MSC微球培养物在软骨培养基中预分化2周，通过提取转化生长因子β (TGF β)、降低地塞米松浓度、添加甲状腺激素（T3）诱导肥大。采用组织学、免疫组织化学和生化方法对培养物进行鉴定，并采用定量逆转录-聚合酶链反应评估基因表达。TGF - β的戒断、地塞米松水平的降低和T3的添加是诱导肥厚的必要条件。细胞形态学变化伴有碱性磷酸酶活性升高、基质矿化和各种肥大标志物的变化，包括X型胶原、成纤维细胞生长因子受体1-3、甲状旁腺激素相关蛋白受体、视黄酸受体γ、基质金属蛋白酶13、印度刺猬蛋白、骨钙素和促凋亡基因p53。然而，在整个培养过程中，肥厚的诱导并不均匀，并且观察到明显的去分化区域。软骨分化MSCs在功能上与生长板软骨细胞相似，表达非常相似的肥厚表型。在这里使用的体外培养条件下，msc衍生的软骨细胞经历了类似于软骨内胚胎骨骼发育过程中观察到的分化程序，具有最终分化的潜力。该培养系统可用于筛选肥大抑制条件和可能有助于增强软骨组织工程中MSC性能的药物。
Objective. Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers.Methods. Human MSC pellet cultures were pre-differentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor beta (TGF beta), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription-polymerase chain reaction.Results. The combination of TGF beta withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1-3, parathyroid hormone-related protein receptor, retinoic acid receptor gamma, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed.Conclusion. Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering.