Jiang Yiyue, Zhang Zheng, Ren Kangkang, Zhou Shujuan, Qiao Yang, Huang Weilu, Zhang Nanyang, Xu Hanyan, Xu Xinping, Wen Bing, Zhang Wei, Yin Lu
Jiangxi Provincial Key Laboratory of Respiratory Diseases, Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, 330209, China.
Jiangxi Hospital of China-Japan Friendship Hospital, Nanchang, Jiangxi, 330209, China.
J Biol Eng. 2025 Jun 3;19(1):52. doi: 10.1186/s13036-025-00524-w.
Dental pulp-derived mesenchymal stromal cells (DPMSCs) represent a promising avenue for regenerative medicine. However, the therapeutic potency of DPMSCs is substantially influenced by their functional heterogeneity emerging during ex vivo expansion. Therefore, identifying and selecting the most potent subpopulation of DPMSCs following expansion is key to improving therapeutic efficacy and consistency. Nevertheless, conventional methods for isolating stem cell subpopulations are largely impractical for clinical-scale bioprocessing, primarily due to their high cost and limited throughput. Recent research has unveiled a strong correlation between the biophysical characteristics of culture-expanded stem cells and their functional attributes, thereby raising the prospect of employing high-throughput, cost-effective biophysical sorting techniques to isolate functionally distinct subpopulations.
A high-throughput microfluidic chip was implemented to perform label-free separation of culture-expanded DPMSC subpopulations based on their varying cell sizes, utilizing the principle of Dean flow fractionation.
Leveraging this microfluidic technology, culture-expanded DPMSCs, isolated from the third molar teeth of adult donors, were fractionated into four subpopulations, each distinguished by distinct average cell diameters ranging from 14.3 to 20.5 μm. The medium-sized subpopulations (15.2 to 18.6 μm) demonstrated the highest colony-forming efficiency. In contrast, the large-sized subpopulation (18.6 to 20.5 μm) showed enhanced osteogenic and chondrogenic potencies in vitro, alongside superior abilities to suppress T cell proliferation and reverse macrophage M1 polarization in different co-culture settings. Furthermore, transcriptomic analysis revealed a progressively shifting gene expression profile with the change in DPMSC size. The large- and medium-sized subpopulation upregulates genes involved in immune responses, calcium signaling, and ECM-receptor interaction, while the small-sized subpopulation downregulates genes associated with immune response pathways, cell cycle, and growth factor activities.
This study elucidates the morphological relevance of the functional heterogeneity of culture-expanded DPMSCs and proposes a viable strategy for the isolation of functional subpopulations of DPMSCs in clinical-scale manufacturing.
The online version contains supplementary material available at 10.1186/s13036-025-00524-w.
牙髓来源的间充质基质细胞（DPMSCs）是再生医学中一条很有前景的途径。然而，DPMSCs的治疗效力在很大程度上受到其在体外扩增过程中出现的功能异质性的影响。因此，在扩增后识别和选择最有效的DPMSCs亚群是提高治疗效果和一致性的关键。然而，传统的分离干细胞亚群的方法在临床规模的生物加工中大多不实用，主要是因为其成本高且通量有限。最近的研究揭示了培养扩增的干细胞的生物物理特性与其功能属性之间存在很强的相关性，从而提出了采用高通量、经济高效的生物物理分选技术来分离功能不同的亚群的可能性。
基于迪恩流分级原理，利用高通量微流控芯片对培养扩增的DPMSC亚群进行基于细胞大小差异的无标记分离。
利用这种微流控技术，从成年供体的第三磨牙中分离出的培养扩增的DPMSCs被分为四个亚群，每个亚群的平均细胞直径不同，范围从14.3到20.5μm。中等大小的亚群（15.2至18.6μm）表现出最高的集落形成效率。相比之下，大尺寸亚群（18.6至20.5μm）在体外显示出增强的成骨和成软骨能力，以及在不同共培养环境中抑制T细胞增殖和逆转巨噬细胞M1极化的卓越能力。此外，转录组分析揭示了随着DPMSC大小的变化，基因表达谱逐渐发生变化。大尺寸和中等尺寸亚群上调参与免疫反应、钙信号传导和细胞外基质受体相互作用的基因，而小尺寸亚群下调与免疫反应途径、细胞周期和生长因子活性相关的基因。
本研究阐明了培养扩增的DPMSCs功能异质性的形态学相关性，并提出了一种在临床规模生产中分离DPMSCs功能亚群的可行策略。
在线版本包含可在10.1186/s13036-025-00524-w获取的补充材料。
