ABSTRACT: Astrogliosis is an inflammatory process by which astrocytes undergo morphological, transcriptional, and functional changes that primarily contribute to tissue healing and restore CNS homeostasis as a response to neuroinflammation. However, excessive astrocyte activation causes neuronal death, immune cell activation and infiltration, and chronic neuroinflammation. The advent of human induced pluripotent stem cell (hiPSC)-derived three-dimensional (3D) models has aided research of complex CNS processes by generating complex structures of neural cells able to decode the molecular mechanisms that trigger and sustain astrocyte activation and drive microenvironment remodeling in the human CNS. Despite depicting interactions between different brain cell types and their microenvironment, the lack of reproducibility, and immature/activated cell phenotypes typical of these models limit their utility to address neuroinflammation mechanisms. Here we establish a hiPSC-derived model of neuroinflammation to dissect cellular crosstalk along the neuroinflammatory axis. A methodology pioneered by our team was used, in which hiPSC-derived neural progenitors were cultured in perfusion stirred-tank bioreactors and differentiated into 3D neurospheroids composed of neurons, astrocytes, and oligodendrocytes. This model recapitulates specific features of the brain microenvironment, such as human brain-like ECM deposition and neuron-glia functional interactions. The neurospheroids were challenged with prototypical inflammatory factors reported to induce activation of astrocytes in mice models, namely TNF-α, IL-α, and C1q. By performing whole transcriptome analysis (RNAseq), we observed an upregulation of several inflammatory-related genes associated with TNF and NF-kB signaling (e.g., CXCL5, CCL2, TNAIP3, and NFKB2). Whole proteome analysis (SWATH-MS) also depicted the enrichment of proteins involved in cell response to stress and cytokine regulation, namely TXLNA, DCTN5, and NOLC1. Concomitantly, time course analysis of conditioned media from the stimulated neurospheroids exhibited increased secretion of inflammation-related cytokines (e.g., CCL2, CXCL10, IL-6, and IL-8). Astrocytes in challenged neurospheroids displayed an impaired capacity for clearance of extracellular glutamate and secretion of glutamine compared to the unstimulated control, indicating functional impairment and known molecular effects of TNF signaling. Together, these results further demonstrate astrocyte functionality within the neurospheroids, which undergo canonical astrogliosis events that are considered hallmarks of neuroinflammation. The human neurospheroid model described may contribute to better understand the mechanisms governing human astrogliosis and be used as a platform to study glial-neuron mature interactions during acute stimulation and inflammation.
