Short and long TNF‐alpha exposure recapitulates canonical astrogliosis events in human‐induced pluripotent stem cells‐derived astrocytes

Abstract

Astrogliosis comprises a variety of changes in astrocytes that occur in a contextspecific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1β, IL-6, and TNF-α in the first hours following TNF-α stimulation.After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNFα-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover,~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction.Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing

TNF Production and Release from Microglia via Extracellular Vesicles: Impact on Brain Functions

“…In addition to the aforementioned effects on microglial cells, TNF released by inflamed microglia, together with IL-1α and C1q, has been demonstrated to drive detrimental A1 astrocyte activation, leading to a final toxic effect on neurons and oligodendrocytes underlying the pathological mechanisms of several neurodegenerative disorders [ 97 ]. Notably, the critical role of TNF in driving astroglial activation has been confirmed also in human astrocytes derived from induced pluripotent stem cells [ 98 ]. Recently, Lombardi and colleagues provided new details on the mechanism driving microglia-induced detrimental astrogliosis, demonstrating that this process is mediated by microglia-derived EVs carrying TNF [ 72 ].…”

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC‐Derived Astrocytes

“…shows the timeline of the protocol until NSCs are obtained. Part 2 is based on a recently published protocol (Trindade et al, 2020). Cells generated using this protocol can show features of astrogliosis when exposed to an inflammatory stimulus (Trindade et al, 2020).…”

“…Please note the typical morphology of differentiated astrocytes in Figure 2. Further assessment can be performed based on immunostaining and functional assays, as described by Trindade et al (2020). 58.…”

“…Astrocytes are also essential players in the regulation of synapses. An imbalance in their function could lead to congenital diseases and neurological disorders (Ledur et al, 2020;Trindade et al, 2020). Despite their utmost importance, astrocytes have been mainly studied in murine systems, and several questions remain regarding astrocytic responses in human cells when compared to other species.…”

“…Recently, we showed that these glial cells can be successfully used to model astrogliosis in vitro. Among typical reactive astrocyte features, we have described the secretion of astrocyte-specific proteins and cytokines after an inflammatory stimulus, TNF-α-mediated nuclear translocation of NF-kB (p65 subunit), and specific morphological changes, as well as altered uptake of aspartate/glutamate (Trindade et al, 2020). Therefore, iPSCderived astrocytes represent an interesting model for studying a range of functions of human astrocytes in health and disease, such as mitochondrial function and stress.…”