■肠胶质细胞是控制运动性的重要角色,肠屏障完整性和炎症。在炎症期间,它们转变为反应性表型,使它们能够释放炎症介质,从而塑造炎症环境。虽然存在大量建立良好的体内模型,破译肠神经胶质反应的机制途径所必需的细胞培养模型的标准化程度较低。特别是,细胞外基质(ECM)的组成会严重影响实验结果。考虑到越来越多的研究涉及原发性肠胶质细胞,需要更好地了解它们的稳态和炎症体外培养条件。
■我们检查了不同的ECM对肠胶质培养物纯度的影响,网络形态和免疫反应性。因此,我们用免疫荧光和明场显微镜,以及3'批量mRNA测序。此外,我们将培养的细胞与从Sox10iCreERT2Rpl22HA/小鼠分离的体内肠神经胶质转录组进行了比较。
■我们认为基质胶和层粘连蛋白优于其他涂层,包括聚-L-鸟氨酸,不同的赖氨酸,胶原蛋白,和纤连蛋白,获得最高的肠神经胶质纯度和表达连接蛋白43半通道的最延伸的神经胶质网络,允许细胞间通讯。转录分析显示,Matrigel和层粘连蛋白上的肠神经胶质细胞具有很强的相似性,并富集了支持神经元分化的基因集,而聚-L-鸟氨酸上的细胞表现出与细胞增殖有关的富集。比较培养的和体内的肠神经胶质转录组显示出50%的重叠,与所用的涂层底物无关。通过IL-1β处理的肠神经胶质的炎症激活显示了不同的涂层依赖性基因表达特征,在基质胶和层粘连蛋白涂层上富集与骨髓和上皮细胞分化相关的基因,而聚-L-鸟氨酸诱导更多与淋巴细胞分化相关的基因集。
■一起,形态的变化,原代肠胶质细胞的分化和免疫激活证明了ECM的强大作用。我们确定了基质胶和层粘连蛋白是鼠肠神经胶质培养物的主要底物。这些新的见解将有助于标准化和提高肠胶质培养质量和未来体外研究之间的可重复性,可以更好地比较它们在肠道神经炎症中的功能作用。
UNASSIGNED: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed.
UNASSIGNED: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3\' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice.
UNASSIGNED: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation.
UNASSIGNED: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.