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A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells
Cellular and Molecular Neurobiology ( IF 4 ) Pub Date : 2021-05-31 , DOI: 10.1007/s10571-021-01109-z
Ekkaphot Khongkla 1 , Kwanchanok Uppakara 2 , Nittaya Boonmuen 1 , Kanit Bhukhai 1 , Witchuda Saengsawang 1, 3, 4
Affiliation  

The Cath.a-differentiated (CAD) cell line is a central nervous system-derived catecholaminergic cell line originating from tyrosine hydroxylase (TH)-producing neurons located around the locus coeruleus area of the mouse brain. CAD cells have been used as an in vitro model for cellular and molecular studies due to their ability to differentiate under serum-free media conditions. However, the lack of serum-derived survival factors, limits the longevity for differentiated CAD cells to be maintained in healthy conditions; thereby, limiting their use in long-term culture studies. Here, we present a novel differentiation method that utilizes dexamethasone (Dex), a synthetic glucocorticoid receptor agonist. Specifically, we discovered that the addition of 100 µM of Dex into the 1% fetal bovine serum (FBS)-supplemented media effectively induced neuronal differentiation of CAD cells, as characterized by neurite formation and elongation. Dex-differentiated CAD cells exited the cell cycle, stopped proliferating, extended the neurites, and expressed neuronal markers. These effects were dependent on the glucocorticoid receptors (GR) as they were abolished by GR knockdown. Importantly, Dex-differentiated CAD cells showed longer survival duration than serum-free differentiated CAD cells. In addition, RNA-sequencing and qPCR data demonstrate that several genes involved in proliferation, neuronal differentiation, and survival pathways were differentially expressed in the Dex-differentiated cells. This is the first study to reveal Dex as a novel differentiation methodology used to generate postmitotic neuronal CAD cells, which may be utilized as an in vitro neuronal model for cellular and molecular neurobiology research.



中文翻译:

一种使用地塞米松诱导中枢神经系统衍生的儿茶酚胺能 CAD 细胞神经元分化的新方法

Cath.a 分化 (CAD) 细胞系是一种源自中枢神经系统的儿茶酚胺能细胞系,源自位于小鼠大脑蓝斑区域周围的产生酪氨酸羟化酶 (TH) 的神经元。CAD 细胞已被用作细胞和分子研究的体外模型,因为它们能够在无血清培养基条件下分化。然而,缺乏血清衍生的存活因子,限制了分化的 CAD 细胞在健康条件下维持的寿命;因此,限制了它们在长期培养研究中的使用。在这里,我们提出了一种利用合成糖皮质激素受体激动剂地塞米松 (Dex) 的新型分化方法。具体来说,我们发现在 1% 胎牛血清 (FBS) 补充培养基中添加 100 µM Dex 可有效诱导 CAD 细胞的神经元分化,其特征在于神经突形成和伸长。Dex 分化的 CAD 细胞退出细胞周期,停止增殖,延长神经突,并表达神经元标志物。这些影响依赖于糖皮质激素受体 (GR),因为它们被 GR 击倒消除了。重要的是,Dex 分化的 CAD 细胞比无血清分化的 CAD 细胞显示出更长的存活时间。此外,RNA 测序和 qPCR 数据表明,参与增殖、神经元分化和存活途径的几个基因在 Dex 分化的细胞中差异表达。

更新日期:2021-05-31
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