The Shapiro Family Laboratory of Viral Oncology and Aging Research, UCLA School of Dentistry, Los Angeles, California.
The Shapiro Family Laboratory of Viral Oncology and Aging Research, UCLA School of Dentistry, Los Angeles, California; Section of Endodontics, UCLA School of Dentistry, Los Angeles, California.
J Endod. 2017 Aug;43(8):1302-1308. doi: 10.1016/j.joen.2017.03.016. Epub 2017 Jun 9.
Mesenchymal stem cells (MSCs) are typically cultured as adherent monolayer using a conventional tissue culture technique. However, this technique incompletely reproduces an in vivo microenvironment of stem cells and results in the loss of stemness properties. Three-dimensional (3D) sphere culture is one of the most widely used 3D culture techniques that have been developed to recapitulate the in vivo microenvironment. However, the stemness and multilineage differentiation capacity of spheres derived from dental pulp stem cells (DPSCs) have not been well investigated.
DPSCs were cultured and examined for the sphere-forming ability in serum-free, nonadherent conditions. The expression of pluripotency transcription factors was assayed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. The expression of MSC-associated markers was determined by flow cytometry. Multilineage differentiation capacity was examined by alkaline phosphatase, alizarin red S, and oil red O assays. Subcutaneous transplantation in nude mice was used to examine the in vivo mineralized tissue-forming ability of sphere and adherent monolayer cells derived from DPSCs.
We showed that DPSCs form spheres. DPSC spheres exhibited a distinct stem cell phenotype characterized by robust expression of pluripotency transcription factors and decreased expression of MSC-associated markers compared with their corresponding adherent monolayer cells. Functionally, DPSC spheres exhibited enhanced in vitro multilineage differentiation capacity. The expression of multilineage differentiation-related genes was also highly increased in DPSC spheres. Furthermore, DPSC sphere cells possessed higher in vivo mineralized tissue-forming ability than adherent monolayer cells.
Our findings indicate that sphere-forming cells are unique multipotent cell populations in DPSCs. Our study further suggests that DPSC spheres may provide a unique opportunity for pulp tissue regeneration.
间充质干细胞（MSCs）通常使用传统的组织培养技术作为贴壁单层进行培养。然而，这种技术不能完全再现干细胞的体内微环境，导致干细胞特性的丧失。三维（3D）球体培养是最广泛使用的 3D 培养技术之一，旨在再现体内微环境。然而，牙髓干细胞（DPSCs）来源的球体的干细胞特性和多能分化能力尚未得到很好的研究。
DPSCs 在无血清、非贴壁条件下培养并检测其球体形成能力。通过逆转录定量聚合酶链反应和 Western blot 分析检测多能转录因子的表达。通过流式细胞术测定 MSC 相关标志物的表达。碱性磷酸酶、茜素红 S 和油红 O 测定法检测多能分化能力。裸鼠皮下移植用于检测 DPSCs 来源的球体和贴壁单层细胞的体内矿化组织形成能力。
我们表明 DPSCs 可以形成球体。与相应的贴壁单层细胞相比，DPSC 球体表现出明显的干细胞表型，具有强大的多能转录因子表达和 MSC 相关标志物表达降低。功能上，DPSC 球体表现出增强的体外多能分化能力。DPSC 球体中多能分化相关基因的表达也显著增加。此外，DPSC 球体细胞比贴壁单层细胞具有更高的体内矿化组织形成能力。
我们的研究结果表明，球体形成细胞是 DPSCs 中独特的多能细胞群体。我们的研究进一步表明，DPSC 球体可能为牙髓组织再生提供了独特的机会。
