Characterization of human dental pulp cells‐derived spheroids in serum‐free medium: Stem cells in the core
Characterization of human dental pulp cells‐derived spheroids in serum‐free medium: Stem cells in the core
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DOI：
10.1002/jcb.24610
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发表时间：
2013-11
影响因子：
4
通讯作者：
Li Xiao;T. Tsutsui
中科院分区：
文献类型：
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作者：
Li Xiao;T. Tsutsui
球体模型导致了对分化、组织组织和动态平衡的更多了解。在本研究中，我们观察到在无血清培养条件下，人牙髓细胞(DPC)自发形成球体，并可存活15周以上。为了表征这些球体，我们研究了它们的动力学、微环境、细胞分布、分子特征和神经元/成骨潜能。细胞示踪实验表明，球体内的细胞周期非常缓慢。虽然球体有缺氧的微环境，但即使经过长期培养，也没有出现任何大规模的细胞死亡。整装免疫荧光染色和组织学分析显示干细胞分布在球体的中央/中间带。QRT-PCR分析表明，茎的标记NANOG、TP63和CD44在球体中的表达明显高于单层培养。神经标志物CDH2、NFM、TUBB3和CD24在球体中的基因表达水平明显高于单层DPC，且随培养时间的延长而增加。无血清培养2周左右，在无神经诱导条件下，球形细胞自发转化为神经标志物Huc/D和P75阳性的神经元样细胞。当球体被转移到成骨介质中时，它们迅速分化为成骨/成牙本质细胞，尤其是中央原始细胞。与单层DPC相比，球体中的矿化显著增加。这种球体模型为探索干细胞稳态和组织结构的分子基础提供了一种研究工具，可广泛用于神经组织和骨再生。J.细胞。生物化学。2013年，114：2624-2636。©2013 Wiley期刊，Inc.
Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum‐free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die‐offs even after long‐term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT‐PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time‐dependent manner. Without any neural induction, spheroid‐derived cells spontaneously converted into neuron‐like cells with positive staining of neural markers HuC/D and P75 under the serum‐free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration. J. Cell. Biochem. 114: 2624–2636, 2013. © 2013 Wiley Periodicals, Inc.