Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Pediatric Dentistry, School of Stomatology, Tongji University, Shanghai 200072, P.R. China.
Shanghai Ninth People's Hospital, Shanghai Jiaotong University, Shanghai 200011, P.R. China.
Mol Med Rep. 2018 May;17(5):6551-6559. doi: 10.3892/mmr.2018.8725. Epub 2018 Mar 9.
Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) are types of human dental tissue‑derived mesenchymal stem cells (MSCs). These cells possess a capacity for self‑renewal, multilineage differentiation potential and immunomodulatory functions. Previous studies have reported that DPSCs and SHED may be beneficial in regenerative treatments and immunotherapy. The substantial expansion of cells in vitro is a prerequisite to obtaining adequate cell numbers required for cell‑based therapy. However, the regeneration and clinical potential of MSCs diminishes with long‑term cell culture amplification. To assess the alterations in SHED and DPSCs characteristics that underlie cellular senescence and result from extended in vitro amplification, the biological properties of SHED and DPSCs at passages 4 (P4) and 20 (P20) were compared. The cells underwent senescence following serial expansion to P20, as determined by altered cell morphology, decreased proliferation and migration capacity, attenuated differentiation potential, elevated senescence‑associated β‑galactosidase (SA‑β‑gal)‑positive rates and increased apoptosis. The phenotypic changes were also accompanied by a marked increase in the expression of p53, p21 and p16Ink4a. The present study also identified that senescent DPSCs exhibited an increased number of positive cells in SA‑β‑gal staining and demonstrated varying expressions of p53, p21 and p16Ink4a in comparison with SHED, indicating the involvement of diverse pathways in cellular senescence during long‑term sequential in vitro culture and passage. Furthermore, at early and late passages, SHED exhibited a higher proliferation rate and osteogenic differentiation capability when compared with DPSCs. In addition, both cell types maintained their characteristic immunophenotype during long‑term cultivation, while the expression levels of CD73 were higher in SHED at P20. The present study concluded that notable alterations were exhibited in SHED and DPSCs during the process of extensive expansion in vitro and the results may provide guidance for the selection of safe and effective expanded SHED and DPSCs for regenerative medicine and therapy.
牙髓干细胞（DPSCs）和人脱落乳牙来源的干细胞（SHED）是人类牙齿组织来源的间充质干细胞（MSCs）的两种类型。这些细胞具有自我更新、多向分化潜能和免疫调节功能。先前的研究表明，DPSCs 和 SHED 可能有益于再生治疗和免疫治疗。体外大量扩增细胞是获得细胞治疗所需足够细胞数量的前提。然而，随着长期细胞培养扩增，MSC 的再生和临床潜力会降低。为了评估导致细胞衰老的 SHED 和 DPSCs 特征变化以及由此产生的细胞体外扩增，比较了 SHED 和 DPSCs 在传代 4（P4）和 20（P20）时的生物学特性。通过改变细胞形态、降低增殖和迁移能力、减弱分化潜能、提高衰老相关β-半乳糖苷酶（SA-β-半乳糖苷）阳性率和增加细胞凋亡，确定细胞在传代至 P20 后发生衰老。表型变化还伴随着 p53、p21 和 p16Ink4a 的表达明显增加。本研究还发现，衰老的 DPSCs 在 SA-β-半乳糖苷染色中显示出更多的阳性细胞，并与 SHED 相比，p53、p21 和 p16Ink4a 的表达也存在差异，表明在长期连续体外培养和传代过程中，细胞衰老涉及不同的途径。此外，在早期和晚期传代时，与 DPSCs 相比，SHED 具有更高的增殖率和成骨分化能力。此外，两种细胞类型在长期培养过程中均保持其特征性免疫表型，而在 P20 时 SHED 的 CD73 表达水平更高。本研究得出结论，在体外广泛扩增过程中，SHED 和 DPSCs 表现出明显的变化，这些结果可能为选择安全有效的扩增 SHED 和 DPSCs 用于再生医学和治疗提供指导。
