Secretome Analysis of Human Mesenchymal Stem Cells Undergoing Chondrogenic Differentiation

Human
mesenchymal stem cells (hMSCs) can be triggered to differentiate
toward chondrocytes and thus harbor great therapeutic potential for
the repair of cartilage defects in osteoarthritis (OA) and other articular
diseases. However, the molecular mechanisms underlying the chondrogenesis
process are still in part unknown. In this work, we followed a double-stable
isotope labeling by amino acids in cell culture (SILAC) strategy to
evaluate the quantitative modulation of the secretome of stem cells
isolated from bone marrow (hBMSCs) during the first steps of their
chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the
identification of 221 proteins with a reported extracellular localization.
Most of them were characteristic of cartilage extracellular matrix,
and 34 showed statistically significant quantitative alterations during
chondrogenesis. These include, among others, cartilage markers such
as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported
markers of cartilage development (Versican), and a suggested marker
of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness
of secretome analysis for understanding the mechanisms responsible
for cartilage matrix formation, and it reports a panel of extracellular
markers potentially useful for the evaluation of tissue development
in cell therapy- or tissue engineering-based approaches for cartilage
repair.

人类间充质干细胞（human mesenchymal stem cells, hMSCs）可被诱导分化为软骨细胞，因此在骨关节炎（osteoarthritis, OA）及其他关节疾病的软骨缺损修复中具备优异的治疗潜力。然而，软骨生成过程背后的分子机制仍有部分尚未阐明。本研究采用细胞培养氨基酸稳定同位素标记（stable isotope labeling by amino acids in cell culture, SILAC）策略，对从骨髓中分离的间充质干细胞（hBMSCs）在软骨生成早期阶段的分泌组定量调控情况进行了评估。通过液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）分析，共鉴定出221种已被报道定位于细胞外的蛋白质。其中绝大多数为软骨细胞外基质的特征性蛋白，另有34种蛋白在软骨生成过程中呈现具有统计学显著性的定量变化。这些蛋白涵盖软骨标志物（如蛋白聚糖4（Proteoglycan 4）、软骨寡聚基质蛋白（COMP））、抗分解代谢标志物（金属蛋白酶组织抑制剂1（TIMP1））、已被报道的软骨发育标志物（Versican），以及一种潜在的软骨生成标志物CRAC1。综上，本研究证实了分泌组分析在解析软骨基质形成相关分子机制中的应用价值，并报道了一组细胞外标志物，其有望用于评估基于细胞治疗或组织工程的软骨修复策略中的组织发育水平。