Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, 2610 Wilrijk, Belgium; Bio-Imaging Lab, University of Antwerp, 2610 Wilrijk, Belgium.
Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, 2610 Wilrijk, Belgium; Cell Death Signaling, Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Wilrijk, Belgium.
Brain Behav Immun. 2024 Oct;121:1-12. doi: 10.1016/j.bbi.2024.07.008. Epub 2024 Jul 17.
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 CXCR1 CCR2 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.
诱导多能干细胞 (iPSC) 衍生的神经球 (NSPH) 模型是一种新兴的体外工具，可用于研究炎症触发因素对 3D 神经环境中神经退行性变和修复的影响。与人类对应物相比，缺乏用于深入表征和验证研究的小鼠 iPSC 衍生的 NSPH 是一个主要的实验研究空白，尽管它们提供了将体外 NSPH 反应与体内大脑反应进行真正比较或验证的唯一可能性。为了促进这些发展，我们在这里描述了 5 周大的 CXCR1 CCR2 小鼠 (m)iPSC 衍生的二部分 (神经元+星形胶质细胞) 和三部分 (神经元+星形胶质细胞+小胶质细胞) NSPH 模型的生成和表征，这些模型可以在受到促炎刺激后进行细胞激活。首先，细胞因子分析表明，两种二部分和三部分 NSPH 在分别用 TNFα+IL1β+IFNγ 和 LPS+IFNγ 刺激 3 天后，都可以被触发释放 IL6 和 CXCL10。此外，对 G3BP1 和 PABPC1 的免疫细胞化学分析表明，在刺激 3 天后，两种二部分和三部分 NSPH 中都形成了应激颗粒。为了进一步研究观察到的炎症反应和细胞应激迹象，我们对稳态和炎症条件下的二部分和三部分 NSPH 进行了非靶向转录组和蛋白质组学分析。在这里，使用未刺激和刺激 NSPH 之间的差异基因和蛋白质表达谱的组合，Ingenuity 通路分析 (IPA) 证实了炎症和细胞应激相关的经典途径在二部分和三部分 NSPH 中被激活。此外，我们的多组学分析表明，与二部分 NSPH 相比，刺激的三部分 NSPH 中下游炎症反应、稳态和发育过程受损以及细胞死亡过程激活的水平更高。总之，这些结果强调了在 NSPH 研究中纳入小胶质细胞的优势，以研究 3D 神经环境中炎症诱导的神经退行性变。
