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. 2024 Aug 1;19(8):1122–1136. doi: 10.1016/j.stemcr.2024.07.002

Induction of astrocyte reactivity promotes neurodegeneration in human pluripotent stem cell models

Cátia Gomes 1,2, Kang-Chieh Huang 2,3, Jade Harkin 2,4, Aaron Baker 1,2, Jason M Hughes 5, Yanling Pan 2,3, Kaylee Tutrow 1,2, Kirstin B VanderWall 2,3, Sailee S Lavekar 2,3, Melody Hernandez 1,2, Theodore R Cummins 2,3, Scott G Canfield 2,5, Jason S Meyer 1,2,4,6,7,8,∗
PMCID: PMC11368677  PMID: 39094561

Summary

Reactive astrocytes are known to exert detrimental effects upon neurons in several neurodegenerative diseases, yet our understanding of how astrocytes promote neurotoxicity remains incomplete, especially in human systems. In this study, we leveraged human pluripotent stem cell (hPSC) models to examine how reactivity alters astrocyte function and mediates neurodegeneration. hPSC-derived astrocytes were induced to a reactive phenotype, at which point they exhibited a hypertrophic profile and increased complement C3 expression. Functionally, reactive astrocytes displayed decreased intracellular calcium, elevated phagocytic capacity, and decreased contribution to the blood-brain barrier. Subsequently, co-culture of reactive astrocytes with a variety of neuronal cell types promoted morphological and functional alterations. Furthermore, when reactivity was induced in astrocytes from patient-specific hPSCs (glaucoma, Alzheimer’s disease, and amyotrophic lateral sclerosis), the reactive state exacerbated astrocytic disease-associated phenotypes. These results demonstrate how reactive astrocytes modulate neurodegeneration, significantly contributing to our understanding of a role for reactive astrocytes in neurodegenerative diseases.

Keywords: astrocyte, reactivity, neurodegeneration, stem cell, disease, differentiation

Highlights

  • •

    hPSC-derived reactive astrocytes exhibit a predominantly inflammatory phenotype

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    Reactive astrocytes promote neurodegenerative features in multiple types of neurons

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    Patient-derived reactive astrocytes result in more robust disease phenotypes

In this article, Gomes and colleagues demonstrate the induction of a proinflammatory reactive phenotype in human pluripotent stem cell-derived astrocytes. Reactive astrocytes contributed to neurodegenerative phenotypes in multiple types of neurons, including retinal ganglion cells, cortical neurons, and motor neurons. Further, when differentiated from cell lines derived from glaucoma, Alzheimer’s disease, and ALS patient samples, the reactive state enhanced disease-associated phenotypes.

Introduction

Astrocytes play important roles in brain homeostasis and neuronal support yet acquire an altered functional profile and contribute to neuroinflammation and neurodegeneration in many disease states (Acioglu et al., 2021). Recently, an astrocyte phenotype that drives neurodegeneration has been identified and characterized, often broadly referred to as neurotoxic reactive astrocytes (Grimaldi et al., 2019; Liddelow et al., 2017; Taylor et al., 2020), yet the underlying neurotoxic mechanisms of reactive astrocytes and their functional consequences upon neurons are not fully understood. Importantly, recent studies have demonstrated the presence of reactive astrocytes in several models of neurodegeneration including Alzheimer’s disease (AD) (Arranz and De Strooper, 2019), retinal injury/glaucoma (Cooper et al., 2018; Guttenplan et al., 2020a), and amyotrophic lateral sclerosis (ALS) (Gomes et al., 2022a; Guttenplan et al., 2020b).

Although animal models serve as powerful models to study neuron-glia interactions, rodents often insufficiently mimic the degenerative state found within patients, and recent studies have demonstrated numerous transcriptional, phenotypic, and functional differences between rodent and primate neurons and glia (Hodge et al., 2019; Oberheim et al., 2009; Zhang et al., 2016), particularly in disease states. Human pluripotent stem cells (hPSCs) constitute a physiologically relevant in vitro human cellular model that overcomes differences between rodent models and human cells (Li et al., 2021; Peng et al., 2019; Zhang et al., 2016). While some recent studies have begun to better characterize reactive astrocytes using hPSC-derived human cellular models (Barbar et al., 2020; Kim et al., 2022; Labib et al., 2022; Leng et al., 2022), the functional consequences of reactive astrocytes upon neurons in a human cellular model remain poorly understood.

To address this shortcoming, we aimed to develop and further characterize properties of hPSC-derived reactive astrocytes in an in vitro human cellular model and assess the effects of these reactive astrocytes upon neurons. Induced reactivity was associated with altered functional properties of hPSC-derived astrocytes, including increased phagocytic capability and decreased contribution to blood-brain barrier (BBB) integrity. When grown in co-culture with a variety of hPSC-derived neurons, reactive astrocytes contributed to the onset of neurodegenerative features. Finally, when reactivity was induced in hPSC-derived astrocytes differentiated from cell lines with patient-specific disease mutations, the reactive state exacerbated the severity of disease-associated phenotypes. Collectively, these results demonstrate that the induction of hPSC-derived astrocytes to a reactive profile alters their functional properties, leading to neurodegenerative features in multiple types of neurons, with these results collectively advancing our understanding of the role of reactive astrocytes in neurodegenerative disease states.

Results

Induction of a reactive phenotype in hPSC-derived astrocytes

A specific phenotype of reactive astrocytes traditionally referred to as neurotoxic reactive astrocytes has been implicated in several neurodegenerative diseases (Grimaldi et al., 2019; Liddelow et al., 2017), and this phenotype can be replicated in vitro through incubation of rodent or hPSC-derived astrocytes with a cocktail of cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), and complement component 1q (C1q) (Barbar et al., 2020; Labib et al., 2022; Liddelow et al., 2017; Matusova et al., 2023; Smith et al., 2022). To further explore this population of astrocytes, we initially sought to identify and characterize specific phenotypes associated with reactivity. Following stimulation with TNF-α, IL-1α, and C1q, hPSC-derived astrocytes exhibited a hypertrophic profile characterized by a more compact appearance and more numerous branches (Figures 1A and 1B). Induced reactive astrocytes also exhibited an increased perinuclear expression of complement factor C3 (Figures 1C and 1D), a specific characteristic of the reactive phenotype (Barbar et al., 2020; Liddelow et al., 2017). Further analyses revealed that reactive astrocytes displayed increased total number and total length of branches (Figures 1E and 1F), while individual branches tended to be shorter and less complex (Figures 1G and 1H). The expression of proteins previously associated with a reactive phenotype were also analyzed by western blot, with a significant increase in the expression of C3, while the expression of GFAP and S100β were unchanged (Figures 1I and 1J). Finally, quantitative reverse-transcription PCR (RT-qPCR) identified a significant upregulation of pan-reactive genes, including TIMP1 and CXCL10, as well as many genes specifically associated with neurotoxic reactive astrocytes, including complement factor C3, GBP2, and PSMB8 (Figure 1K). To ensure the reproducibility of this phenotype across hPSC lines (Brunner et al., 2023; Volpato and Webber, 2020), all results were independently replicated using three unrelated lines of hPSCs throughout all studies.

Figure 1.

Figure 1

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Induction of a reactive phenotype in hPSC-derived astrocytes

(A and B) Reactive astrocytes exhibited a hypertrophic profile as seen by S100β and GFAP staining.

(C and D) Increased accumulation of C3 staining in the perinuclear region of reactive astrocytes.

(E–H) Morphological analysis showed an increased number of branches and shorter processes in reactive astrocytes, as well as an overall increased number of process intersections through Sholl analysis.

(I and J) Increased C3 expression exhibited by reactive astrocytes through western blot analysis.

(K) qPCR analysis showed that reactive astrocytes express increased levels of pan-reactive associated genes, as well as genes that specifically characterize the phenotype of neurotoxic reactive astrocytes. Results are represented as fold change vs. control astrocytes in (J) and (K). Colored dots correspond to 3 different lines, H7 hPSC line in blue, mips2 hPSC line in gray, and WTC11 hPSC line in orange. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. control samples, two-tailed paired Student’s t test. Scale bar: 30 μm in (A) and (B), 20 μm in (C) and (D). Data represents mean values ± SEM.

We then sought to further characterize the transcriptional profile of hPSC-derived astrocytes in the induced reactive state. We confirmed that, after incubation with the cocktail of reactive astrocyte-inducing cytokines, astrocytes exhibited an upregulation of several genes linked to neuroinflammation (Figures 2A and 2B), including complement C3 and nuclear factor κB (NF-κB), which are known to be involved in inflammatory responses (Liddelow et al., 2017; Smith et al., 2022). Additionally, we observed upregulation of genes such as SQSTM1 and optineurin (OPTN), with known roles in autophagy, the latter of which has been associated with neurodegenerative diseases including glaucoma and ALS. Subsequent pathway enrichment analyses identified numerous cellular pathways differentially modulated due to the induction of a reactive phenotype, including a downregulation of genes linked to the regulation of apoptotic processes and programmed cell death, as well as an upregulation of genes linked to cytokine signaling and inflammatory pathways (Figures 2C and 2D). These results support the establishment of a specific reactive phenotype in hPSC-astrocytes, further supporting their use to study the contribution of reactive astrocytes to neurodegeneration.

Figure 2.

Figure 2

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Transcriptional analysis of reactive hPSC-derived astrocytes compared to controls

(A) Heatmap representing differentially expressed genes in reactive and control astrocytes. The up- and downregulated genes are represented as red and blue colored, respectively.

(B) Volcano plot exhibiting differentially expressed genes in reactive astrocytes compared to controls. Significantly upregulated genes are shown in blue, while significantly downregulated genes are shown in green.

(C and D) GO and pathway analysis based on differential gene expression of reactive and control astrocytes.

Induction of a reactive state in hPSC-astrocytes modulates functional outcomes

As astrocytes exhibit many functional changes and lose neurosupportive roles upon the acquisition of a reactive state (Phatnani and Maniatis, 2015; Sofroniew and Vinters, 2010), we explored whether functional properties of astrocytes were affected after induction to reactivity. To study phagocytic capacity, both control and reactive astrocytes were incubated with pHrodo Red E. coli BioParticles for 48 h, with images taken and analyzed by time-lapse imaging every 4 h (Video S1, Figures 3A and 3B) and results demonstrating that reactive astrocytes have an increased phagocytic capacity (Figure 3C). Additionally, since astrocytic calcium is an important modulator of synaptic transmission and other cellular processes (Guerra-Gomes et al., 2017; Shigetomi et al., 2019), we found that reactive astrocytes displayed decreased levels of intracellular calcium exhibited by decreased fluorescence of Fluo-4 AM (Figures 3D–3F). Moreover, as astrocytes also modulate neuronal function through paracrine mechanisms (Ramírez-Jarquín et al., 2017; Tripathi et al., 2017), we analyzed conditioned media using the Meso Scale Discovery V-PLEX proinflammatory panel, in which reactive astrocytes secreted significantly increased levels of nearly all analyzed inflammatory soluble factors (Figure 3G).

Figure 3.

Figure 3

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Functional changes in hPSC-derived astrocytes following induction of reactivity

(A and B) Representative images of pHrodo particles engulfed by control and reactive astrocytes.

(C) The percentage of astrocytes that had engulfed pHrodo particles significantly increased from 16 to 48 h.

(D–F) Representative images of Fluo-4 AM-positive astrocytes following stimulation with ATP, and (F) quantification demonstrating a significant decrease in fluorescence intensity in reactive astrocytes compared to control cells. ANOVA followed by Tukey’s multiple comparison test or by two-tailed paired Student’s t test, in (C) and (F), respectively.

(G) Meso Scale Discovery analysis showed an increased secretion of inflammatory factors by reactive astrocytes compared to control astrocytes. Two-tailed unpaired Student’s t test with Welch’s correction. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Data represent mean values ±SEM. Scale bar represents 100 μm in (B) and 30 μm in (E).

Video S1. Timelapse video recording demonstrating the phagocytosis of pHrodo bioparticles by control and reactive astrocytes. Phagocytosed pHrodo bioparticles fluoresce red after uptake by astrocytes
Download video file (67.4MB, mp4)

Astrocytes are also important contributors to BBB homeostasis, while reactive astrocytes lead to BBB disruption and increased permeability (Kim et al., 2022). To study whether hPSC-derived reactive astrocytes modulate BBB properties, transwell co-cultures were established between astrocytes and brain microvascular endothelial cells (BMECs) (Canfield et al., 2017). We first confirmed the expression of endothelial cell-associated markers in hPSC-derived BMEC cultures (Figures 4A–4F). We then observed that reactive astrocytes induced lower transendothelial electrical resistance (TEER) levels in BMECs (Figure 4G), while also promoting a significant increase in permeability (Figure 4H). We then investigated P-glycoprotein (PGP) efflux transporter activity by measuring the transport of rhodamine 123 across the BMEC monolayer. An increased transport of rhodamine 123 following cyclosporine A (CsA) inhibition was observed in BMECs co-cultured with control astrocytes, indicative of PGP activity in induced pluripotent stem cell (iPSC)-BMECs, while reactive astrocytes exhibited a decreased transport of rhodamine 123 (Figure 4I). Overall, these results suggested that the induction of the reactive phenotype in hPSC-astrocytes leads to important alterations in astrocyte function that might be linked to a neurotoxic or lack of neurosupportive role for these glial cells observed in neurodegeneration.

Figure 4.

Figure 4

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Reactive hPSC-derived astrocytes contribute to blood-brain barrier disruption

Co-cultures were performed using a transwell system, with brain microvascular endothelial cells (BMECs) seeded on the transwell and astrocytes seeded in the bottom of the well.

(A–I) Representative images of iPSC-derived BMECs expressing characteristic endothelial cell markers, including Claudin-5, glucose transporter 1 (GLUT-1), Occludin, and zonula occludens 1 (ZO-1). Reactive astrocytes induced a reduction in transendothelial electrical resistance (TEER) levels (G), increased fluorescein permeability (H), and elevated rhodamine 123 transport (I), compared to the effect promoted by control astrocytes. CsA, cyclosporin. ANOVA followed by Tukey’s multiple comparison test in (G) and (H) and by Šídák’s multiple comparisons test in (I). Data represent mean values ±SEM. ∗p < 0.05 and ∗∗p < 0.01, BMEC monoculture vs. BMECs co-cultured with control astrocytes; ###p < 0.001, BMEC monoculture vs. BMECs co-cultured with reactive astrocytes; &&p < 0.01, BMECs co-cultured with control astrocytes vs. BMECs co-cultured with reactive astrocytes in (G). ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 in (H) and (I). Scale bar: 50 μm.

Reactive hPSC-derived astrocytes promote neuronal morphological alterations and contribute to neurodegeneration

We next studied whether activated astrocytes could promote neurodegenerative phenotypes by establishing co-culture systems between control or reactive astrocytes with hPSC-derived RGCs (Harkin et al., 2024; Ohlemacher et al., 2016), the cell type primarily affected in optic neuropathies such as glaucoma (Figure 5). In this context, RGCs were either in direct contact with astrocytes (representative images in Figure S1) or exposed to paracrine signals in transwell conditioned medium models. While effects of reactive astrocytes were observed within 1 week of co-culture (Figure S2), stronger effects were observed after 2 weeks of co-culture (Figures 5A–5G), with a significant reduction in the number of primary neurites, total neurite length, and overall neurite arborization. Next, whole-cell patch-clamp recordings demonstrated that RGCs grown with control astrocytes fired more spontaneous action potentials overall (Figure 5H), while RGCs grown with reactive astrocytes were more easily excitable in response to a delivered depolarizing current, including both a significantly increased frequency of action potential firing and an increase in the total number of action potentials observed (Figures 5I–5K). These changes in response to reactive astrocytes were also associated with a significant decrease in the action potential current threshold and capacitance of RGCs (Figures 5L and 5M). Finally, to determine if the cytokine cocktail used to induce astrocytic reactivity influenced neurons on their own, RGCs were incubated alone with TNF-α, IL-1α, and C1q for 1 and 2 weeks (Figure S3), with results confirming that, in the absence of astrocytes, the cocktail of cytokines did not affect neuronal morphology.

Figure 5.

Figure 5

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hPSC-derived reactive astrocytes induce retinal ganglion cell neurotoxicity and hyperexcitability

Astrocytes were co-cultured with RGCs, in either direct-contact or transwell cultures and induced to a reactive state through incubation with IL-1α, TNF-α, and C1q for 2 weeks. Representative images of RGCs (BRN3b:tdTomato) co-cultured with control or reactive astrocytes, in direct co-cultures (A and B), or transwell cultures (C and D).

(E and F) Reactive astrocytes promoted a reduction in the number of primary neurites and total neurite extension in RGCs, in both direct-contact and transwell culture systems.

(G) The overall complexity of RGC neurite outgrowth, assessed by Sholl analysis, was significantly decreased in both direct-contact and transwell culture systems. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 in (E) and (F). ∗∗∗∗p < 0.0001, RGCs co-cultured with control astrocytes vs. reactive astrocytes in contact-dependent cultures, and ####p < 0.0001, RGCs co-cultured with control astrocytes vs. reactive astrocytes in transwell cultures, in (G). One-Way ANOVA followed by Šídák’s multiple comparisons test with selected pair.

(H–J) Upon whole-cell patch-clamp recording, RGCs were less likely to be spontaneously active in co-culture with reactive astrocytes compared to controls yet fired more action potentials upon delivery of a depolarizing current.

(K–M) RGCs co-cultured with reactive astrocytes had a higher frequency of action potentials fired, while they also exhibited a significantly lower action potential current threshold and capacitance. ∗p < 0.05 and ∗∗∗∗p < 0.0001. Scale bar: 100 μm. Data represent mean values ± SEM.

Next, we sought to determine if similar toxic effects of reactive astrocytes could be observed with other neuronal cell types. To accomplish this, we differentiated hPSCs into cortical neurons and spinal motor neurons (Figure 6A), characteristic of the neuronal cell types affected in diseases such as AD and ALS, respectively, and co-cultured them with astrocytes (Figure S1). Reactive astrocytes promoted a reduction in the number of primary neurites and neurite length, as well as in the overall neurite complexity upon both cortical neurons (Figures 6B–6H) and motor neurons (Figures 6I–6O), although secreted factors were not able to reduce the total number of primary neurites in hPSC-derived motor neurons. Interestingly, cortical neurons seemed to be more resistant to the effects promoted by reactive astrocytes, since toxic effects were observed only after 2 weeks of co-culture (Figures 6B–6H and S2). Altogether, these results suggested that reactive astrocytes rely on both contact-dependent and soluble factors to induce neurodegeneration of different neuronal types and that the magnitude of these effects can vary based upon the neuronal cell type.

Figure 6.

Figure 6

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hPSC-derived reactive astrocytes also induce neurotoxic phenotypes upon cortical neurons and motor neurons

(A) Schematic demonstrating the derivation of both cortical neurons and motor neurons from hPSCs, with resulting cortical neurons identified by CTIP2 and MAP2 expression, while motor neurons could be identified by HB9 and βIII-tubulin expression. Images in Panel A created with BioRender.com.

(B–H) Cortical neurons were grown either in direct contact with astrocytes (B and C) or in transwell models (D and E). After two weeks of co-culture, reactive astrocytes resulted in a significantly decreased number of primary neurites from cortical neurons (F), a decrease in total neurite length (G), as well as in overall morphological complexity by Sholl analysis (H).

(I–O) Similarly, motor neurons were grown either in direct contact with astrocytes (I and J) or in transwell models (K and L). Morphological analyses demonstrated that reactive astrocytes promoted a reduction in the number of primary neurites in motor neurons, only in direct-contact culture system (M), as well as a significant reduction in total neurite length (N) and overall outgrowth complexity (O). Data represent mean values ±SEM from at least four independent experiments per hPSC line. One-Way ANOVA followed by Šídák’s multiple comparisons test with selected pair; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and n.s. means non-significant, in (F), (G), (M), and (N). ∗∗∗∗p < 0.0001, cortical neurons (in H) or motor neurons (in O) co-cultured with control astrocytes vs. reactive astrocytes in contact-dependent cultures, and ####p < 0.0001, cortical neurons (in H) or motor neurons (in O) co-cultured with control astrocytes vs. reactive astrocytes in transwell cultures. Scale bar: 100 μm.

Induction of a reactive state in disease-associated astrocytes exacerbates disease-specific features

To study the acquisition of the neurotoxic reactive phenotype in astrocytes associated with specific neurodegenerative diseases, we sought to derive astrocytes from hPSCs with varied neurodegenerative-associated gene mutations, including glaucoma (OPTN-E50K), AD (PSEN1-N141I), and ALS (SOD1-N139K). Following differentiation, all three cell lines robustly gave rise to GFAP-expressing astrocytes (Figures 7A–7F). Subsequently, after incubation with TNF-α, IL-1α, and C1q, all OPTN, PSEN1, and SOD1 hPSC-derived astrocytes exhibited a hypertrophic profile with numerous and thicker branches (Figures 7A–7F), as well as a strong upregulation of complement C3 (Figures 7G–7L and S4). Reactive astrocytes with a glaucoma OPTN-E50K mutation displayed more numerous yet shorter branches, and an overall increased number of intersections across increasing distance from the soma (Figures 7M–7O). Next, as OPTN functions as an autophagy receptor, and deficits in the autophagy pathway have been associated with the OPTN-E50K mutation (Gomes et al., 2022b; Ying and Yue, 2016), we found that reactive OPTN-E50K astrocytes displayed further increased expression of P62 as well as LC3-II (Figure 7P), suggesting that the induction of a reactive phenotype exacerbates autophagy dysfunction and disease phenotypes in glaucoma OPTN-E50K astrocytes.

Figure 7.

Figure 7

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Induction of a reactive phenotype exacerbates disease-associated phenotypes in patient-specific cell lines

Astrocytes were differentiated from hPSC lines carrying mutations associated with either glaucoma OPTN(E50K), Alzheimer’s disease (PSEN1-N141I), or ALS (SOD1-N139K).

(A–L) Upon induction of reactivity, astrocytes from all backgrounds developed a hypertrophic profile (A–F) and increased accumulation of C3 (G–L). Among astrocytes with the glaucoma OPTN-E50K mutation (M–O), reactivity led to a significant increase in the number of branches, along with a significant decrease in longest branch length as well as morphological complexity by Sholl analysis.

(P) Induction of reactivity also exacerbated disease-related phenotypes such as a significant increase in autophagy proteins P62 and the LC3-II/I ratio.

(Q–S) Similarly, among astrocytes with the Alzheimer’s PSEN1-N141I mutation, reactivity led to a significant increase in the number of branches along with a significant decrease in longest branch length, although no significant differences were observed in overall morphological complexity.

(T–U) Astrocyte reactivity led to a decreased capacity for amyloid beta uptake compared to homeostatic PSEN1 astrocytes.

(V–X) ALS SOD1-N139K astrocytes induced to reactivity exhibited an increased number of branches along with a significant decrease in the longest branch length, although no differences were observed in overall morphological complexity.

(Y and Z) Reactive SOD1-N139K astrocytes exhibited more prominent aggregates of SOD1 protein and had significantly increased intracellular levels of SOD1. Data represent mean values ±SEM, two-tailed paired Student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. respective control astrocytes. Scale bar: 30 μm.

Next, we sought to determine if a reactive state could also modulate disease phenotypes in astrocytes with an AD gene mutation. Similar to glaucoma astrocytes, those with an AD PSEN1-N141I mutation exhibited more numerous yet shorter branches, although a Sholl analysis of branching complexity did not identify any differences in overall morphological complexity (Figures 7Q–7S). As increased accumulation and aggregation of amyloid-β (Aβ) are hallmark features of AD (Selkoe and Hardy, 2016), we found a decrease in the uptake of Aβ1-42 in reactive PSEN1 astrocytes (Figures 7T and 7U), suggesting that the induction of a reactive phenotype in PSEN1 astrocytes compromises their capacity to phagocytose Aβ1-42. Finally, we then sought to determine a role for reactivity in modulating astrocyte disease phenotypes in ALS using an SOD1-N139K hPSC line, in which a reactive phenotype led to significantly more numerous yet shorter branches, although no other morphological differences were observed in overall complexity (Figures 7V–7X). As SOD1 protein aggregation has been observed in ALS patient glial cells (Forsberg et al., 2010), SOD1 aggregation was increased in the perinuclear region in reactive SOD1 hPSC-derived astrocytes (Figure 7Y and 7Y′). Moreover, an increased level of total SOD1 protein was also observed in reactive SOD1 astrocytes (Figure 7Z). Overall, these results suggested that the induction of a reactive phenotype in astrocytes carrying specific genetic mutations associated with neurodegenerative disorders can exacerbate phenotypes and characteristics of the disease state.

Discussion

The presence of a reactive astrocyte phenotype across several neurodegenerative diseases (Grimaldi et al., 2019; Liddelow et al., 2017) underscores the importance of understanding the phenotypic and functional changes that accompany the induction of reactivity in human astrocytes. In the present study, the induction of the reactive phenotype was performed in astrocytes derived from three unrelated hPSC lines to ensure the reproducibility of the astrocytic reactive phenotype across different genetic backgrounds (Brunner et al., 2023; Volpato and Webber, 2020). Interestingly, previous studies have shown that reactive astrocytes exhibit shorter branches, and a consequent reduction in their spatial coverage, which potentially leads to a detachment of astrocytic processes that might be associated with axonal survival (Sun et al., 2009, 2010). Therefore, the process shortening observed in hPSC-derived astrocytes after induction to a reactive phenotype may suggest a loss of neurosupportive roles.

Although classically considered a marker for glial reactivity, the lack of evident upregulation after induction of the neurotoxic reactive phenotype may further suggest that GFAP may not be the best marker to access reactivity. Indeed, previous studies have shown decreased GFAP expression in some disease-associated astrocytes (Diaz-Amarilla et al., 2011; Gomes et al., 2019). Increased expression of complement C3 was also confirmed in reactive hPSC-astrocytes as a specific marker of this neurotoxic phenotype (Liddelow et al., 2017), and transcriptional data also revealed other differentially expressed genes associated with the complement cascade, including SERPING1 and SERPINA3, suggesting a further role for the complement pathway in astrocyte reactivity. Similarly, both SQSTM1 and OPTN were upregulated in reactive astrocytes, further suggesting a prominent role for autophagy. Further, genes such as PINK1 and MAPT, whose variants have been associated with Parkinson’s disease and frontotemporal dementia, respectively, were both found to be misregulated in the reactive astrocyte state, suggesting a role for astrocyte reactivity and inflammation in disease states.

Transcriptional analyses revealed that reactive hPSC-astrocytes expressed some markers previously related to a neuroprotective reactive phenotype (e.g., CLCF1 and PTX3), while others were downregulated (S100A10 and B3GNT5) (Liddelow et al., 2017). The discrepancy in the expression of these markers may be caused by astrocyte heterogeneity (Escartin et al., 2021), especially when linked to glial reactivity (Anderson et al., 2014; Matusova et al., 2023), suggesting that the subcategorization of reactive phenotypes of astrocytes might be limiting when assessing neurotoxic populations of these glial cells. Interestingly, hPSC-derived astrocytes also upregulated several genes that characterize the inflammatory reactive astrocyte signature identified by Leng et al. (2022), also supporting an inflammatory profile in our model of reactive astrocytes, as well as a potential role in neurodegeneration.

Functionally, reactive hPSC-derived astrocytes showed increased phagocytic capacity, which has previously been described in reactive astrocytes after brain injury (Morizawa et al., 2017) and perhaps works as a compensatory mechanism when microglia are dysfunctional (Konishi et al., 2020). However, decreased phagocytic capacity was recently described in CD49f+ hPSC-derived reactive astrocytes (Barbar et al., 2020). This discrepancy may result from the different type of particles used and uptake analysis, or because these authors focused on a specific subpopulation of CD49f+ cells. Moreover, decreased intracellular calcium was observed in reactive hPSC-derived astrocytes, and decreased calcium signaling has been observed in rodent models of AD before amyloid plaque deposition (Shah et al., 2022). Aberrant calcium signaling in astrocytes has also been associated with neuronal dysfunction and neurodegeneration (Bancroft and Srinivasan, 2021), with our results further corroborating the idea that reactive astrocytes act to disrupt BBB integrity as well (Cabezas et al., 2014; Liu et al., 2018).

The consequences of astrocyte reactivity on neuronal morphology and function in human cells remain unclear and scarcely explored, despite the fact that they have been identified in several neurodegenerative diseases (Anderson et al., 2014; Gomes et al., 2022a; Liddelow and Barres, 2017). Our results suggest a prevailing neurotoxic effect by reactive astrocytes across neuronal cell types, although it was intriguing that cortical neurons seemed to be more resilient to the neurotoxic effects elicited by reactive astrocytes compared to RGCs and motor neurons, as significant differences were not observed until later stages. These experiments also demonstrated that reactive astrocytes could induce neurotoxicity by paracrine signaling without cell-to-cell contact. Future studies will focus on the identification of the soluble factors that mediate this effect, which could greatly contribute to the development of novel therapeutic strategies for neurodegenerative diseases characterized by a strong reactive astrocytic phenotype.

When reactivity was induced in hPSC-derived astrocytes differentiated from cell lines obtained from a variety of neurodegenerative disease patient samples, the reactive state exacerbated the magnitude of disease-associated phenotypes. The induction of the reactive state in OPTN-E50K astrocytes increased the expression of autophagy-related markers, suggesting an exacerbation of autophagy dysfunction typically associated with this mutation (Gomes et al., 2022b; Sung and Jimenez-Sanchez, 2020). Moreover, the induction of reactivity in PSEN1 astrocytes compromised their capacity to phagocytose Aβ1-42, in line with prior studies showing that, in AD, reactive and inflammatory astrocytes are found around Aβ plaques (Gomez-Arboledas et al., 2018; Serrano-Pozo et al., 2016), and previous studies described a similar reduced phagocytic capacity in astrocytes derived from ApoE4 hPSCs (Lin et al., 2018). Finally, the increased aggregation of SOD1 protein observed in SOD1-N139K hPSC-derived reactive astrocytes is in line with previous studies showing SOD1 aggregates in ALS patient glial cells (Forsberg et al., 2010). Collectively, our studies showed that the induction of a reactive state in hPSC-derived astrocytes carrying genetic mutations linked to neurodegenerative diseases better mimics phenotypes observed in patient tissue, constituting a powerful human cellular model to study mechanisms of disease.

Taken together, the results of this study provide a comprehensive assessment of features associated with human astrocyte reactivity, as well as how reactive astrocytes modulate neuronal phenotypes and function. More so, our results demonstrate an important role for astrocyte reactivity related to the in vitro modeling of neurodegenerative diseases, with the induction of reactivity shown to exacerbate disease-associated phenotypes in astrocytes, significantly contributing to our understanding of the role for reactive astrocytes in neurodegenerative diseases.

Experimental procedures

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jason Meyer (meyerjas@iu.edu).

Materials availability

There were no new reagents generated for this study.

Data and code availability

All relevant data from this study are available within the article and supplemental information. Transcriptional data from control and reactive astrocytes analyzed by the NanoString platform can be found in the supplemental files.

Maintenance of hPSCs

hPSCs were maintained and expanded using established procedures as previously described (Fligor et al., 2020) and detailed in the supplemental information.

Astrocyte differentiation and induction of reactivity

hPSCs were differentiated to an astrocytic lineage according to previously established protocols (Gomes et al., 2022b; Krencik and Zhang, 2011), and as described in the supplemental information. To induce a reactive phenotype, hPSC-derived astrocytes were incubated with TNF-α (30 ng/mL, PeproTech), IL-1α (3 ng/mL, PeproTech), and C1q (400 ng/mL, MyBioSource) for 48 h, as previously described (Liddelow et al., 2017).

Neuronal differentiation and establishment of co-cultures

hPSCs were differentiated into RGCs (VanderWall et al., 2020), cortical neurons (Pankratz et al., 2007), and motor neurons (Li et al., 2005), using previously published protocols. Detailed methods for the differentiation of each neuronal cell type are available in the supplemental information. To establish co-cultures between astrocytes and each of the aforementioned neuron cell types, co-cultures were generated by combining neurons and astrocytes at a ratio of 1:2. These co-cultures were maintained in BrainPhys medium (STEMCELL Technologies) for 2 weeks for neuronal maturation before addition of cytokines. After these 2 weeks, TNF-α, IL-1α, and C1q were freshly added every three days to maintain astrocyte reactivity for up to additional two weeks.

Immunocytochemistry

Cells were plated onto poly-ornithine and laminin-coated coverslips, fixed with 4% paraformaldehyde, and immunostained as previously described (Gomes et al., 2022b). Detailed procedures are provided in the supplemental information as well as Table S1.

RT-qPCR

RT-qPCR experiments were performed as previously described (Meyer et al., 2011). Detailed methods, including primer sequences, can be found in the supplemental information as well as Table S2.

nCounter neuroinflammation panel analysis

Gene expression profiling of control and reactive astrocytes was accomplished using the nCounter human neuroinflammation panel on the nCounter platform (NanoString), as described by the manufacturer and detailed in the supplemental information.

Western blot

Protein expression was determined by western blot as described before (Gomes et al., 2022b), and detailed in the supplemental information.

Meso Scale Discovery assay

To assess secreted factors from both control and reactive astrocytes, we examined conditioned medium via the Meso Scale Discovery V-PLEX proinflammatory panel, as we have previously described (Gomes et al., 2022b). More detailed methods can be found in the supplemental information.

Calcium imaging

To assess differences in intracellular calcium, astrocytes were incubated with the calcium-sensitive dye Fluo-4 AM and fluorescence intensity was analyzed after stimulation with ATP, with results represented as ΔF/F0. Detailed procedures are provided in the supplemental information.

Phagocytosis assays

To determine changes in the ability of reactive astrocytes to phagocytose material, both control and reactive astrocytes were incubated with pHrodo Red E. coli BioParticles and imaged every 4 h for 48 h. In other experiments, aggregated Aβ1-42 was then added to astrocytes for 48 h. Detailed procedures are provided in the supplemental information.

BBB-related analysis

To determine changes in the ability of reactive astrocytes to contribute to BBB integrity compared to control astrocytes, we used an established transwell barrier model according to previously established protocols (Canfield et al., 2017). BMECs were differentiated from iPSCs, and transwell co-cultures were established with either control or reactive astrocytes. Measurements of the TEER, barrier permeability to sodium fluorescein, and PGP efflux transporter activity were made. Detailed procedures are provided in the supplemental information.

Statistical analyses

Detailed description of the statistical analyses performed for each result is available in the supplemental information.

Acknowledgments

We thank AJ Baucum for assistance with calcium imaging experiments, Emily Welby and Allison Ebert for sharing the SOD1 hPSC line, Adrian Oblak and Audrey Lee-Gosselin for their help with the NanoString experiments, as well as Peter Lin for assistance with Meso Scale Discovery assays. Grant support was provided by the National Eye Institute (R01EY033022 and U24EY033269 to J.S.M.), the National Institute of Neurological Disorders and Stroke (R01NS053422 to T.R.C.), the BrightFocus Foundation (G2020369 to J.S.M.), the Gilbert Family Foundation (923016 to J.S.M.), the Glaucoma Research Foundation (to J.S.M.), and the Indiana Department of Health Spinal Cord and Brain Injury Research Fund (26343 to J.S.M.). Support for this project was also provided by the Sarah Roush Memorial Fellowship from the Indiana Alzheimer’s Disease Center (C.G.) and the BrightFocus Postdoctoral Fellowship (G2022003F to C.G.), as well as a Cagiantas scholarship from the Indiana University School of Medicine (J.H.). This publication was also made possible with partial support from the Stark Neurosciences Research Institute/Eli Lilly and Co. Neurodegeneration fellowship (K.-C.H.). This publication was also made possible, in part, with support from the Indiana Clinical and Translational Sciences Institute Collaboration in Translational Research Pilot Grant (to J.S.M., T.R.C., and S.G.C.) funded, in part, by grant number UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical & Translational Sciences Award.

Author contributions

C.G., T.R.C., S.G.C., and J.S.M. designed the research. C.G., K.-C.H., J.H., J.M.H., Y.P., A.B., K.T., S.S.L., and M.H. performed the research. C.G., K.-C.H., J.M.H., Y.P., T.R.C., S.G.C., and J.S.M. analyzed the data. C.G., S.G.C., and J.S.M. wrote the paper.

Declaration of interests

J.S.M. holds a patent related to methods for the retinal differentiation of human pluripotent stem cells used in this study.

Published: August 1, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.07.002.

Supplemental information

Document S1. Figures S1–S5 and Tables S1–S3
mmc1.pdf (3.1MB, pdf)
Document S2. Article plus supplemental information
mmc3.pdf (9.7MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Timelapse video recording demonstrating the phagocytosis of pHrodo bioparticles by control and reactive astrocytes. Phagocytosed pHrodo bioparticles fluoresce red after uptake by astrocytes
Download video file (67.4MB, mp4)
Document S1. Figures S1–S5 and Tables S1–S3
mmc1.pdf (3.1MB, pdf)
Document S2. Article plus supplemental information
mmc3.pdf (9.7MB, pdf)

Data Availability Statement

All relevant data from this study are available within the article and supplemental information. Transcriptional data from control and reactive astrocytes analyzed by the NanoString platform can be found in the supplemental files.

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