Human Induced Pluripotent Stem Cell-Derived Astrocytes Are Differentially Activated by Multiple Sclerosis-Associated Cytokines

Abstract

SummaryRecent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

“…18,19 Where CNTF-reactive astrocytes are predominantly found in the white matter, 20 FBS administration is standardly used to maintain cortical astrocytes, 21 leading to a flat morphology that is common for gray matter astrocytes in culture. However, we lack iPSC-based differentiation protocols generating specific subtypes.…”

“…CNTF and FBS are both used in astrocyte differentiation protocols. 18,19 Where CNTF-reactive astrocytes are predominantly found in the white matter, 20 FBS administration is standardly used to maintain cortical astrocytes, 21 leading to a flat morphology that is common for gray matter astrocytes in culture. Here we used CNTF-and FBS-based media to generate mouse WM astrocytes and mouse GM astrocytes, respectively.…”

CD49f is a novel marker to purify functional human iPSC-derived astrocytes

“…The advent of human embryonic stem cell and induced pluripotent stem cell (iPSC) technology has enabled large-scale generation of human astrocytes and other CNS cells that retain the genetic information of the patient, as powerful human in vitro models of disease (reviewed in 14 ) . Several protocols to generate astrocytes have been developed by independent groups, using either a specific gradient of patterning agents to mimic embryonic development [15][16][17][18][19][20][21] or overexpression of critical transcription factors 22,23 . Usually, iPSC differentiation into astrocytes in monolayer cultures does not require a purification step; in recent years, however, more complex 3D cultures of CNS organoids have been established that contain neural progenitor cells, neurons, oligodendrocyte lineage cells and microglia, in addition to astrocytes 22,[24][25][26][27][28][29][30][31] .…”

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

“…Both GFAP and Vimentin are considered markers of astrocytes and their overexpression characterizes primary features of reactive astrogliosis, since their deletion reduces reactive gliosis and promotes synaptic regeneration (Wilhelmsson et al, ). TNF‐α is fairly known to induce overexpression of GFAP and Vimentin (Perriot et al, ; Zhang et al, ) and our human iPSC‐derived astrocytes were responsive to long‐term TNF‐α exposure by increasing GFAP and Vimentin. The long‐term exposure to TNF‐α was also able to alter astrocyte morphology in vitro, since they displayed an elongated shape, namely polarized astrocytes, resembling those in response to a wound (Peng & Carbonetto, ) or recruited to mold the glial scar around injured sites (Adams & Gallo, ).…”

“…In order to provide not only a characterization of human astrocyte‐secreted proteins following inflammatory stimulus, we also evaluated and characterized major events related to the time course of astrogliosis, such as: (a) TNF‐α‐mediated nuclear translocation of NF‐kB p65 subunit, (b) increases in gene expression and secretion of inflammation‐related cytokines, (c) alterations in cell morphology and shrinkage of astrocyte nuclei, and (d) dysfunctional aspartate/glutamate uptake. Although human iPSC‐derived astrocytes have been shown to respond to inflammatory stimuli (Perriot et al, ; Santos et al, ), it was unclear whether human iPSC‐derived astrocytes can reproduce in vitro the temporally ordered physiological changes typical of astrogliopathology.…”