Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, Florida.
University of Münster, Münster, Germany.
Biotechnol Bioeng. 2020 Oct;117(10):3136-3149. doi: 10.1002/bit.27474. Epub 2020 Jul 5.
Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three-dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation-based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.
蛋白质动态平衡对于细胞功能至关重要，因为动态平衡的丧失与衰老和不需要的蛋白质积累有关。人类间充质干细胞（MSCs）由于其出色的分泌炎症调节剂、血管生成和再生细胞因子的能力，显示出有希望的治疗潜力。然而，存在着人类 MSC 扩增而治疗质量受损的问题。在体外扩增过程中，人类 MSCs 被种植在坚硬的塑料上，并经历培养适应，这导致异常增殖、代谢变化和自噬活性降低。先前已经表明，三维（3D）聚集可以通过增强自噬并将代谢状态恢复到原始表型来逆转其中一些改变。为了进一步了解人类 MSC 培养中的蛋白质动态平衡，本研究调查了 3D 聚集对人类 MSC 蛋白质组的影响，以确定聚集改变的特定途径。分析了源自骨髓（bMSC）和脂肪组织（ASC）的 3D 聚集物和 2D 培养物以及分化的人类真皮成纤维细胞（FB）。蛋白质组学分析显示，3D 聚集物中真核起始因子 2 途径升高，整合应激反应（ISR）的活性上调。特定蛋白质的定量进一步确定 bMSC 和 ASC 对 ISR 有反应，而 FB 没有。3D 聚集显著提高了 bMSC 和 ASC 的缺血存活能力。小分子 salubrinal 和 GSK2606414 对 ISR 的干扰导致 bMSC、ASC 和 FB 产生不同的反应。本研究表明，基于聚集的预处理培养通过建立 ISR 和动态平衡，有可能提高扩增的人类 MSCs 的治疗效果。
