Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons
Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons
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DOI：
10.1016/j.neuint.2009.03.017
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发表时间：
2009-10-01
影响因子：
4.2
通讯作者：
Varga, Gabor
中科院分区：
文献类型：
--
作者：
Kiraly, Marianna;Porcsalmy, Balazs;Varga, Gabor
牙髓干细胞（DPSC）的可塑性已被几项研究所证实，这些研究表明，它们似乎可以通过几次传代进行自我维持，从而产生各种细胞。本研究的目的是在体外将DPSC分化为显示电压门控离子通道活性的功能证据的成熟神经元细胞。首先，将DPSC培养物接种在聚-L-赖氨酸包被的表面上，并用含有碱性成纤维细胞生长因子和脱甲基化剂5-氮杂胞苷的培养基预处理48小时。然后通过同时激活蛋白激酶C和环磷酸腺苷途径进行神经诱导。最后，通过用神经营养素-3、二丁酰环AMP和其他补充成分连续处理来实现诱导细胞的成熟。未诱导的DPSC已经表达波形蛋白、巢蛋白、N-微管蛋白、神经生成素-2和neurogenin-M。诱导处理导致波形蛋白、巢蛋白、N-微管蛋白的表达减少，而神经元生成素-2、神经元特异性烯醇化酶、神经元特异性M和胶质细胞酸性蛋白的表达增加。到成熟期结束时，除了波形蛋白和巢蛋白外，所有研究的基因都以高于未分化对照的水平表达。膜片钳分析揭示了分化细胞中电压依赖性钠和钾通道的功能活性。我们的结果表明，虽然大多数存活的细胞显示神经元形态并表达神经元标志物，但通过本文所述的体外分化方案获得的分化细胞之间存在功能异质性。然而，这项研究清楚地表明，牙髓含有一个细胞群，能够通过我们的三步神经诱导方案进行神经定型。(C)2009爱思唯尔有限公司保留所有权利。
The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-L-lysine coated surfaces and pretreated for 48 h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this Study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol. (C) 2009 Elsevier Ltd. All rights reserved.