School of Stomatology, Qingdao University, Qingdao, 266003, China.
Qingdao Engineering Vocational College, Qingdao, 266000, China.
Biomed Eng Online. 2024 Oct 18;23(1):102. doi: 10.1186/s12938-024-01298-w.
BACKGROUND/PURPOSE: The use of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques is a significant area of global research. The objective of this study was to investigate the effects of long-term in vitro culture on the multidifferentiation potential of hDPSCs and the potential molecular mechanisms involved.
The tissue block method was used to extract hDPSCs from orthodontic-minus-extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages three, six, nine, and twelve were selected. Flow cytometry was used to detect the expression of stem cell surface markers, and CCK-8 was used to assess cell proliferation. β-Galactosidase staining was employed to detect cellular senescence, Alizarin Red S staining to assess osteogenic potential, and Oil Red O staining to evaluate lipogenic capacity. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes in DPSCs and investigate their potential mechanisms.
With increasing passage numbers, pulp stem cells showed an increase in senescence and a decrease in proliferative capacity and osteogenic-lipogenic multidifferentiation potential. The expression of stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90, and CD105 decreased with increasing passages. According to the RNA-seq analysis, the differentially expressed genes CFH, WNT16, HSD17B2, IDI1, and COL5A3 may be associated with stem cell senescence.
Increased in vitro expansion induced cellular senescence in pulp stem cells, which resulted in a reduction in their proliferative capacity and osteogenic-lipogenic differentiation potential. The differential expression of genes such as CFH, WNT16, HSD17B2, IDI1, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.
背景/目的：人牙髓干细胞（hDPSCs）作为组织修复和再生技术的自体干细胞的应用是全球研究的一个重要领域。本研究的目的是探讨长期体外培养对 hDPSCs 多向分化潜能的影响及其潜在的分子机制。
采用组织块法从正畸减数拔牙患者中提取 hDPSCs，体外扩增培养 12 代。选取第 3、6、9、12 代的干细胞。采用流式细胞术检测干细胞表面标志物的表达，CCK-8 法检测细胞增殖情况。β-半乳糖苷酶染色检测细胞衰老，茜素红 S 染色评估成骨潜能，油红 O 染色评估脂生成能力。进行 RNA 测序（RNA-seq）以鉴定 DPSCs 中的差异表达基因，并探讨其潜在机制。
随着传代次数的增加，牙髓干细胞衰老增加，增殖能力和向成骨-成脂多向分化潜能下降。干细胞表面标志物 CD34 和 CD45 的表达稳定，而 CD73、CD90 和 CD105 的表达随传代次数的增加而降低。根据 RNA-seq 分析，差异表达基因 CFH、WNT16、HSD17B2、IDI1 和 COL5A3 可能与干细胞衰老有关。
体外扩增诱导牙髓干细胞衰老，导致其增殖能力和向成骨-成脂分化潜能下降。CFH、WNT16、HSD17B2、IDI1 和 COL5A3 等基因的差异表达可能代表牙髓干细胞衰老的潜在机制。
