Abstract：
induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1β + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.
摘要：
诱导多能干细胞（iPSC）衍生的神经球状体（NSPH）模型是一种新兴的体外工具包，用于研究炎症触发因素对3D神经环境中神经变性和修复的影响。与它们的人类对应物相反，缺乏鼠iPSC衍生的NSPHs进行深入的表征和验证研究是一个主要的实验研究空白，即使它们提供了真正比较或验证体外NSPH反应与体内脑反应的唯一可能性。为了促进这些发展，我们在此描述了5周龄CX3CR1eGFP+/-CCR2RFP+/-鼠(m)iPSC衍生的二分(神经元+星形胶质细胞)和三分(神经元+星形胶质细胞+小胶质细胞)NSPH模型的产生和表征,这些模型可以在促炎性刺激后进行细胞激活.首先,细胞因子分析表明，在刺激三天后，可以触发双向和三方NSPHs释放IL6和CXCL10，分别,TNFα+IL1β+IFNγ和LPS+IFNγ。此外,对G3BP1和PABPC1的免疫细胞化学分析显示，刺激3天后，两部分和三部分NSPH中的应激颗粒均发育。为了进一步研究观察到的炎症反应和细胞应激的迹象，我们在稳态和炎症条件下对二部和三部NSPHs进行了非靶向转录组和蛋白质组分析.这里,使用未刺激和刺激的NSPHs之间的组合差异基因和蛋白质表达谱，独创性通路分析(IPA)证实了在二分和三分NSPH中与炎症和细胞应激相关的典型通路的激活。此外，我们的多组学分析表明下游炎症反应水平较高，体内平衡和发育过程受损，以及与双向NSPH相比，刺激的三方NSPH中细胞死亡过程的激活。Concluding,这些结果强调了将小胶质细胞纳入NSPH研究以研究3D神经环境中炎症诱导的神经变性的优势.
