Juneja Deppo S, Nasuto Slawomir, Delivopoulos Evangelos
School of Biological Sciences, University of Reading, Reading, United Kingdom.
Front Cell Neurosci. 2020 Jan 21;13:579. doi: 10.3389/fncel.2019.00579. eCollection 2019.
Astrocytes are multifunctional cells in the CNS, involved in the regulation of neurovascular coupling, the modulation of electrolytes, and the cycling of neurotransmitters at synapses. Induction of astrocytes from stem cells remains a largely underdeveloped area, as current protocols are time consuming, lack granularity in astrocytic subtype generation, and often are not as efficient as neural induction methods. In this paper we present an efficient method to differentiate astrocytes from mouse embryonic stem cells. Our technique uses a cell suspension protocol to produce embryoid bodies (EBs) that are neurally inducted and seeded onto laminin coated surfaces. Plated EBs attach to the surface and release migrating cells to their surrounding environment, which are further inducted into the astrocytic lineage, through an optimized, heparin-based media. Characterization and functional assessment of the cells consists of immunofluorescent labeling for specific astrocytic proteins and sensitivity to adenosine triphosphate (ATP) stimulation. Our experimental results show that even at the earliest stages of the protocol, cells are positive for astrocytic markers (GFAP, ALDH1L1, S100β, and GLAST) with variant expression patterns and purinergic receptors (P2Y). Generated astrocytes also exhibit differential Ca transients upon stimulation with ATP, which evolve over the differentiation period. Metabotropic purinoceptors P2YR are expressed and we offer preliminary evidence that metabotropic purinoceptors contribute to Ca transients. Our protocol is simple, efficient and fast, facilitating its use in multiple investigations, particularly studies of engineered neural networks.
星形胶质细胞是中枢神经系统中的多功能细胞，参与神经血管耦合的调节、电解质的调节以及突触处神经递质的循环。从干细胞诱导生成星形胶质细胞在很大程度上仍是一个未充分发展的领域，因为目前的方案耗时，在星形胶质细胞亚型生成方面缺乏精细度，而且通常不如神经诱导方法有效。在本文中，我们提出了一种从小鼠胚胎干细胞分化出星形胶质细胞的有效方法。我们的技术采用细胞悬浮方案来产生经神经诱导的胚状体（EBs），并将其接种到层粘连蛋白包被的表面。接种的EBs附着在表面，并向周围环境释放迁移细胞，这些细胞通过优化的基于肝素的培养基进一步被诱导进入星形胶质细胞谱系。对细胞的表征和功能评估包括对特定星形胶质细胞蛋白的免疫荧光标记以及对三磷酸腺苷（ATP）刺激的敏感性。我们的实验结果表明，即使在方案的最早阶段，细胞对星形胶质细胞标志物（胶质纤维酸性蛋白、醛脱氢酶1L1、S100β和谷氨酸转运体1）呈阳性，具有不同的表达模式和嘌呤能受体（P2Y）。生成的星形胶质细胞在受到ATP刺激时也表现出不同的钙瞬变，这种瞬变在分化过程中会发生变化。代谢型嘌呤受体P2YR表达，我们提供了初步证据表明代谢型嘌呤受体促成了钙瞬变。我们的方案简单、高效且快速，便于在多项研究中使用，特别是在工程神经网络的研究中。
