Lee Hsiang-Tzu, Fu Sheng-Yung, Weng Wei-Han, Chao Wei-Chen, Hsu Yu-Pao, Yang Nan-Ping, Chen Yu-Hsu, Chen Shau-Kwaun, Chang Chien-Wen
Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 300044, Taiwan.
Department of Orthopedic Surgery, Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, Taiwan.
Drug Deliv Transl Res. 2025 Sep 6. doi: 10.1007/s13346-025-01927-4.
The three-dimensional (3D) culture system has emerged as an indispensable platform for modulating stem cell function in biomedicine, drug screening, and cell therapy. Despite a few studies confirming the functionality of 3D culture, the molecular factors underlying this process remain obscure. Here, we have utilized a hanging drop method to generate 3D spheroid-derived mesenchymal stem cells (3D MSCs) and compared them to conventionally 2D-cultured MSCs. The results showed that 3D MSCs exhibited distinct phenotypic features than 2D-cultured MSCs and expressed different transcriptional responses, as found from RNA-Seq analysis. Gene ontology (GO) annotations and KEGG pathway mapping pinpointed that the 3D MSCs responded more actively to incoming signals as they upregulated receptors and cytokine production while downregulating proteolysis-, cytoskeletal-, extracellular matrix-, and adhesion-related genes. Functionally, these MSCs also displayed enhanced chemotaxis and improved pulmonary transgression post-intravenous injection. This study provides mechanistic insights on addressing a significant limitation of MSC therapy: pulmonary entrapment after systemic delivery. Moreover, upregulated pluripotency-associated genes, such as Oct4, Sox2, and Nanog, suggest that the 3D MSCs possessed enhanced stemness and regenerative capacity. The results indicate that 3D spheroid culture reshapes MSC transcriptomic and functional profiles and emerges as a promising strategy for improving their therapeutic potential in regenerative medicine.
三维（3D）培养系统已成为生物医学、药物筛选和细胞治疗中调节干细胞功能不可或缺的平台。尽管有一些研究证实了3D培养的功能，但这一过程背后的分子因素仍不清楚。在这里，我们利用悬滴法生成3D球体来源的间充质干细胞（3D MSCs），并将它们与传统二维培养的MSCs进行比较。结果表明，3D MSCs表现出与二维培养的MSCs不同的表型特征，并表现出不同的转录反应，这是通过RNA测序分析发现的。基因本体（GO）注释和KEGG通路映射指出，3D MSCs对传入信号反应更积极，因为它们上调了受体和细胞因子的产生，同时下调了蛋白水解、细胞骨架、细胞外基质和粘附相关基因。在功能上，这些MSCs在静脉注射后还表现出增强的趋化性和改善的肺迁移能力。这项研究为解决MSC治疗的一个重大局限性：全身给药后的肺滞留提供了机制上的见解。此外，多能性相关基因如Oct4、Sox2和Nanog的上调表明3D MSCs具有增强的干性和再生能力。结果表明，3D球体培养重塑了MSCs的转录组和功能谱，并成为提高其在再生医学中治疗潜力的一种有前景的策略。
