Tian Linghan, Wang Weibin, Li Xuzhen, Chen Yan, Song Qian, Yuan Lu, Hao Tingting, Gu Jiaming, Dong Jian
Department of Yunnan Tumor Research Institute, Kunming, 650118, China.
The Third Affiliated Hospital of Kunming Medical University, Kunming, 650118, China.
Heliyon. 2024 Mar 12;10(6):e27996. doi: 10.1016/j.heliyon.2024.e27996. eCollection 2024 Mar 30.
Human umbilical cord mesenchymal stem cells (UC-MSCs) are one of the most extensively researched stem cell types due to their potential for multi-lineage differentiation, secretion of regenerative factors, modulations of immunological activities, and the release of regenerative substances and influence immunological processes. Since UC-MSCs must be cultivated on a large scale for clinical use, selecting the appropriate storing passage, such as the usage-based passage of UC-MSCs, is critical for long-term autologous or allogeneic usage. Long-term cultivation of stem cells, on the other hand, causes them to lose their pluripotent differentiation capacity. As a result, distinguishing between high and low passages of UC-MSCs and identifying the particular variations associated with stem cells and their modes of action is essential for regenerative medicine. Therefore, we investigated the biological features and transcriptional changes of UC-MSCs over passages.
UC-MSCs were isolated from the tissues of the human umbilical cord, and UC-MSCs from five passages (P1, P3, P5, P10 and P15) with three repetitions were compared and identified based on morphology, cell markers, differentiation capacity, and aging-related characteristics. It was previously assumed that the phenotype of cells before the P10 passage was stable, defined as early passage, and that culture could be continued until the 15th passage, defined as late passage. Next, the five passages of UC-MSCs were sequenced using high-throughput complete transcriptome sequencing. Fuzzy C-Means Clustering (FCM) and Weighted Gene Co-expression Network Analysis (WGCNA) were used to find hub genes, and gene silencing was performed to investigate the impact of missing genes on the stemness of UC-MSC cells.
UC-MSCs of different passages displayed similar surface markers, including , , , , and . However, the proliferation time of late-phase UC-MSCs was longer than that of early-phase UC-MSCs, and the expression of the senescence-associated (SA)-β-gal staining marker was higher. At the same time, pluripotency markers (, , and ) were down-regulated, and the multi-differentiation potential was reduced. Meanwhile, and were down-regulated in late-phase UC-MSCs, which were involved in the maintenance of stemness.
and were highly expressed in early-UC-MSCs and showed a downward gradient trend with cell expansion . They regulated UC-MSC proliferation, colony sphere formation, multiple differentiation, stemness maintenance, and other biological manifestations. Therefore, they are anticipated to be new biomarkers for UC-MSCs quality identification in regenerative medicine applications.
人脐带间充质干细胞（UC-MSCs）因其多向分化潜能、分泌再生因子、调节免疫活性以及释放再生物质并影响免疫过程的能力，成为研究最为广泛的干细胞类型之一。由于UC-MSCs必须大规模培养以供临床使用，选择合适的储存传代，如基于使用情况的UC-MSCs传代，对于长期自体或异体使用至关重要。另一方面，干细胞的长期培养会导致其失去多能分化能力。因此，区分UC-MSCs的高代和低代，并确定与干细胞及其作用模式相关的特定差异，对于再生医学至关重要。因此，我们研究了UC-MSCs传代过程中的生物学特性和转录变化。
从人脐带组织中分离出UC-MSCs，并对来自五个传代（P1、P3、P5、P10和P15）且各有三个重复的UC-MSCs，基于形态学、细胞标志物、分化能力和衰老相关特征进行比较和鉴定。此前认为P10代之前细胞的表型稳定，定义为早期传代，培养可继续至第15代，定义为晚期传代。接下来，使用高通量全转录组测序对UC-MSCs的五个传代进行测序。采用模糊C均值聚类（FCM）和加权基因共表达网络分析（WGCNA）来寻找枢纽基因，并进行基因沉默以研究缺失基因对UC-MSC细胞干性的影响。
不同传代的UC-MSCs表现出相似的表面标志物，包括 、 、 、 、 和 。然而，晚期UC-MSCs的增殖时间比早期UC-MSCs长，衰老相关（SA）-β-半乳糖苷染色标志物的表达更高。同时，多能性标志物（ 、 、 和 ）下调，多分化潜能降低。此外，晚期UC-MSCs中参与干性维持的 和 下调。
和 在早期UC-MSCs中高表达，并随着细胞传代呈下降梯度趋势。它们调节UC-MSC的增殖、集落球形成、多向分化、干性维持及其他生物学表现。因此，它们有望成为再生医学应用中UC-MSCs质量鉴定的新生物标志物。
