Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville, Victoria, Australia.
Br J Pharmacol. 2009 Dec;158(8):1922-31. doi: 10.1111/j.1476-5381.2009.00479.x.
Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs.
mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies.
Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors.
GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation.
源自小鼠胚胎干细胞（mESCs）的神经元是基础药理学研究的宝贵资源。除了心肌细胞之外，对于干细胞衍生的分化细胞的药理学知之甚少。在这项研究中，我们研究了 P2 受体激动剂对源自 mESCs 的 GABA 能神经元的作用。
在 N2B27 培养基中，将 mESCs 分化为 GABA 能神经元。在分化的第 24 天，使用钙成像和[3H]-GABA 释放研究来研究 GABA 能神经元对嘌呤能激动剂的反应。
亚群的 GABA 能神经元对一些或所有腺嘌呤和尿嘧啶核苷酸 ATP、ADP、UTP 和 UDP（均为 100μM）作出反应，引起细胞内 Ca2+浓度升高([Ca2+]i)。对 ATP 作出反应的神经元数量减少suramin（100μM）、PPADS（10μM）和 MRS2179（10μM），但不减少 NF023（10μM）。ATP 的反应受细胞外 Zn2+和 pH 的调节。神经元也对 ATP（100μM）作出反应，释放[3H]-GABA，该效应完全被河豚毒素（100nM）抑制。Ap4A 和 2-甲基硫代 ATP 均引起明显的[3H]-GABA 释放。逆转录 PCR 显示存在 P2X1、2、3、4、5、6 和 P2X7，以及 P2Y1、2 和 P2Y6 受体。mESCs 表达 P2X2、5 和 P2X7 以及 P2Y1、2 和 P2Y6 受体。
源自干细胞的 GABA 能神经元主要通过激活 P2X2、P2X4 和 P2Y1 受体来升高[Ca2+]i。这项研究表明，mESCs 产生了用于体外药理学研究的良好神经元功能模型。
