Introduction

Cell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture.

Methods

DPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction.

Results

In proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions.

Conclusions

Our result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

中文翻译：

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在无异源/无血清条件下鉴定人牙髓干细胞产生的神经球。

介绍

基于细胞的疗法需要使用容易收获的细胞来源的新兴替代疗法。来自各种组织的神经干细胞，包括脑、骨髓、皮肤和视网膜，可以产生神经元和神经胶质细胞。最近，人类牙髓干细胞 (DPSCs) 和来自人类脱落乳牙 (SHED) 的干细胞被证明具有间充质干细胞样能力，例如自我更新和多向分化，包括神经元和神经胶质细胞。此外，与成人骨髓基质干细胞相比，DPSCs 和 SHED 表现出更高的增殖率和更高的群体倍增数。因此，DPSCs 是一种有用的来源，可用于各种神经系统疾病的细胞替代疗法。通常，DPSCs 的常规培养方法使用血清，因此，培养基中未定义的成分可能会使控制 DPSCs 自我更新和分化的分子机制的研究复杂化。然而，神经干细胞在悬浮液中增殖形成“神经球”在存在表皮生长因子 (EGF) 和碱性成纤维细胞生长因子 (bFGF) 的情况下进行体外试验。迄今为止，还没有研究在原代培养的无血清条件下从 DPSCs 中获得神经球。因此，本研究的目的是建立一种在原代培养中异种和无血清条件下 DPSCs 增殖和神经分化的方法。

方法

DPSCs 从健康个体（18-41 岁）的智齿牙髓中获得，并在含有 15% 胎牛血清和无异种/无血清培养基的常规培养基中培养。我们通过流式细胞术、免疫组织化学和实时聚合酶链反应评估了异种/无血清条件下 DPSCs 的增殖、神经球的产生和神经分化。

结果

在没有异种/血清的增殖培养基中，DPSCs可以增殖并产生神经球，但是，神经球的自我更新能力有限。在分化条件下，III 类 β-微管蛋白 (TUBB3) 和微管相关蛋白 (MAP2) 在异种/无血清培养条件下的 DPSCs 衍生的神经球中比常规培养条件下的 DPSCs 中更显着地表达。

结论

我们的结果表明，在异种/无血清培养中从 DPSCs 生成神经球可能是神经元退行性疾病临床细胞替代疗法的可及来源。