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Astrocyte Progenitors Derived From Patients With Alzheimer Disease Do Not Impair Stroke Recovery in Mice

Originally publishedhttps://doi.org/10.1161/STROKEAHA.122.039700Stroke. 2022;53:3192–3201

Abstract

Background:

Species-specific differences in astrocytes and their Alzheimer disease-associated pathology may influence cellular responses to other insults. Herein, human glial chimeric mice were generated to evaluate how Alzheimer disease predisposing genetic background in human astrocytes contributes to behavioral outcome and brain pathology after cortical photothrombotic ischemia.

Methods:

Neonatal (P0) immunodeficient mice of both sexes were transplanted with induced pluripotent stem cell–derived astrocyte progenitors from Alzheimer disease patients carrying PSEN1 exon 9 deletion (PSEN1 ΔE9), with isogenic controls, with cells from a healthy donor, or with mouse astrocytes or vehicle. After 14 months, a photothrombotic lesion was produced with Rose Bengal in the motor cortex. Behavior was assessed before ischemia and 1 and 4 weeks after the induction of stroke, followed by tissue perfusion for histology.

Results:

Open field, cylinder, and grid-walking tests showed a persistent locomotor and sensorimotor impairment after ischemia and female mice had larger infarct sizes; yet, these were not affected by astrocytes with PSEN1 ΔE9 background. Staining for human nuclear antigen confirmed that human cells successfully engrafted throughout the mouse brain. However, only a small number of human cells were positive for astrocytic marker GFAP (glial fibrillary acidic protein), mostly located in the corpus callosum and retaining complex human-specific morphology with longer processes compared with host counterparts. While host astrocytes formed the glial scar, human astrocytes were scattered in small numbers close to the lesion boundary. Aβ (beta-amyloid) deposits were not present in PSEN1 ΔE9 astrocyte-transplanted mice.

Conclusions:

Transplanted human cells survived and distributed widely in the host brain but had no impact on severity of ischemic damage after cortical photothrombosis in chimeric mice. Only a small number of transplanted human astrocytes acquired GFAP-positive glial phenotype or migrated toward the ischemic lesion forming glial scar. PSEN1 ΔE9 astrocytes did not impair behavioral recovery after experimental stroke.

Astrocytes play a central role in normal brain function by regulating blood flow, synaptic function and plasticity, as well as maintaining balance of extracellular ions, fluids, and transmitters.1 In response to cerebral insults such as stroke, astrocytes undergo a complex process called reactive astrogliosis characterized by hypertrophy, proliferation, and scar formation.2,3 Given that the changes in astrocytes are long lasting in the perilesional cortex critical to brain reorganization and in turn functional recovery, astrogliosis could result in beneficial and/or harmful consequences.

Species-specific differences make it challenging to study the contribution of astrocytes to the stroke recovery process. Human astrocytes are more numerous,4 and the phenotypes of human cortical astrocytes are more complex and diverse than in their rodent counterparts.5 Regarding stroke, human astrocytes exhibit greater susceptibility to oxidative stress compared with mouse astrocytes, due to the differences in mitochondrial physiology and detoxification pathways.6 In addition, different signaling pathways in astrocytes are activated in response to hypoxia and inflammation between human and mouse. Because of species-specific differences in astrocytes, ischemic pathology and recovery processes are likely to be different in experimental animals and patients, which has major implications in translational research.

About 14% of patients with stroke have preexisting mild cognitive impairment or dementia.7 Preexisting Alzheimer disease (AD) increases the risk of hemorrhagic stroke8 and mortality after stroke.9–11 Experimental studies have confirmed that coexistence of AD pathology and stroke leads to exacerbated outcomes,12–14 possibly through activation of glial cells and upregulation of inflammatory mediators.15 Moreover, induced pluripotent stem cell (iPSC)-derived astrocyte progenitors from AD patients carrying AD predisposing PSEN1 deletion in exon 9 (PSEN1 ΔE9) manifest hallmarks of disease pathology, including increased β-amyloid (Aβ) production, altered cytokine release, and dysregulated Ca2+ homeostasis,16 which all may exaggerate possible ischemic pathology.

Human glial chimeric mice offer a unique model to study how human astrocytes contribute to disease pathogenesis.17–19 Mice transplanted with iPSC-derived astrocyte progenitors generated from patients with amyotrophic lateral sclerosis18 and schizophrenia20,21 show disease-related abnormal behavior suggesting an important role of astrocytes in disease progression. Here, we explored whether human iPSC-derived astrocyte progenitors transplanted in immunodeficient mice contribute to severity of stroke-related pathology and functional impairments after cortical photothrombosis. We hypothesized (1) that human astrocytes impact spontaneous sensorimotor recovery and (2) that PSEN1 ΔE9-related astrocyte malfunctions lead to more severe ischemic damage and behavioral impairment.

Methods

This article adheres to the American Heart Association Journal implementation of the Transparency and Openness Promotion. Comprehensive details for all methods are provided in the Supplemental Material. The datasets generated during the current study are available from the corresponding author on reasonable request.

All animal procedures were approved by the Animal Ethics Committee (Hämeenlinna, Finland), and conducted in accordance with the guidelines set by the European Community Council Directives 86/609/EEC. Altogether, 3 previously established iPSC lines were used in this study as approved by the committee on Research Ethics of Northern Savo Hospital district (license no. 123/2016).

Immunodeficient Rag1tm1Mom mutant mouse (The Jackson Laboratory) pups were transplanted on postnatal day 0 (P0) with human iPSC-astrocyte progenitors derived from AD patients carrying PSEN1 ΔE9 (hAD), with isogenic controls (iCTRL), with a healthy donor astrocytes (hCTRL), or mouse astrocytes or were injected with vehicle (mCTRL). The health of mice was monitored by weight every third month and daily for food and water intake, general assessment of animal activity, and fur condition. At the age of 14 months, cortical ischemia was produced by intravenous Rose Bengal and cold light exposure. Behavioral outcome was assessed using behavioral tests sensitive for motor activity, sensorimotor performance, and gait before ischemia, 1 week after ischemia, and at the end of the 4 weeks follow-up. After the follow-up, the mice were perfused for histology and stained for host and human astrocytes (Figure 1A). The exclusion criteria were decided before the experiment to include (1) welfare problems before ischemia induction (eg, injuries due to aggressive behavior; n=10), (2) failure in transplantation or low cell survival based on missing human nuclei (HuNu)-positive cells in the cortex (n=5), and (3) no lesion based on histology (n=10). The final numbers of mice in experimental groups were the following: hAD (n=19), iCTRL (n=11), hCTRL (n=19), and mCTRL (n=17).

Figure 1.

Figure 1. Characterization and transplantation of human astrocyte progenitors. Study design (A). Representative bright field image of astrocytes matured for 7 d and fluorescence staining for GFAP (glial fibrillary acidic protein) and GFAP/AQP4 (aquaporin4). Nuclei are stained with4',6-diamidino-2-phenylindole (DAPI) (B). Scale bar=50 μm. Part A was created with www.BioRender.com.

Rigor study criteria were followed. For cell transplantation, randomization was not possible, but the cells were prepared separately for each day in random order. If the litter consisted of more than 9 pups, the litter was split in 2 and injected with 2 cell batches. Both female and male mice were included in the study, since at time of transplantation, sex of pups was not known. Behavioral testing was done on separate days for males and females. Surgery, all behavioral analysis, and histology were carried out in a blinded manner.

Results

iPSCs Differentiate Into Astrocytes

Correct differentiation of iPSC-derived astrocyte progenitors was analyzed immunohistochemically to confirm that the majority of cells were positive for astrocyte markers GFAP (glial fibrillary acidic protein) and/or AQP4 (aquaporin4) before transplantation (Figure 1B).

Differences in Body Weight, Infarct Size, and Number of Transplanted Cells

The welfare of mice was followed carefully after cell transplantation, and atypical behavior was not observed. Body weight was measured every third month and there was a significant overall group effect (P<0.01; Figure S1A). The mCTRL group gained more weight compared with hAD (P<0.001) and hCTRL (P<0.001) mice. In addition, male mice gained more weight than females (P<0.001; Figure S1B), and there was a group × sex interaction (P<0.05), indicating that weight gain was different between males and females within groups.

Photothrombosis was selected as it is recommended for long-term stroke recovery studies by the Stroke Recovery and Rehabilitation Roundtable Translational Working Group.22 Induction of photothrombosis by intravenous Rose Bengal injection produced limited damage in the motor cortex, not affecting the corpus callosum (Figure 2A). After excluding mice with no lesion, infarct size was not different between groups (Figure 2B), yet female mice had overall larger ischemic damage (P<0.05).

Figure 2.

Figure 2. Infarct size and human nuclei (HuNu)-positive cells in mice transplanted with hiPSC-derived astrocyte progenitors. Typical lesion location in the motor cortex from Nissl-stained section for a male (1.58 mm3) and female (1.69 mm3) mouse (A). Infarct size measured after behavioral follow-up was not different among experimental groups (B) but was larger in female mice (red symbols [color online only]) compared with males (black symbols). HuNu-positive cells were counted from the motor cortex and were absent in mouse controls (mCTRL) but present in the human iPSC-astrocyte progenitors derived from AD patients (hAD), isogenic control astrocytes (iCTRL), and healthy donor control (hCTRL) groups (C). Cell numbers were higher in ipsilateral side (D). Values are mean±SD. Scale bar=20 μm (C). *P<0.05 compared with contralateral and ipsilateral side; #P<0.05 compared with hCTRL.

Successful cell transplantation was assessed by counting HuNu-positive cells in the cortex (Figure 2C and 2D). No HuNu-positive cells were detected in mCTRL mice. The number of HuNu-positive cells in the ipsilateral cortex (lesion side) was higher in mice transplanted with hAD or iCTRL compared with mice transplanted with hCTRL cells (P<0.05). The number of HuNu-positive cells in the ipsilateral cortex was significantly higher compared with the contralateral cortex in the hAD group (P<0.05).

While HuNu-positive cells were detected widely in the brain, the cell type marker profile and identity of cells was variable. Some HuNu cells double positive with hGFAP (human glial fibrillary acidic protein) were detected in the corpus callosum, with PDGFRα (platelet-derived growth factor receptor alpha), a marker for oligodendrocytes in the cortex, and with doublecortin (DCX), a marker for newborn neurons in the subventricular zone (Figure S2).

Locomotor Activity Was Decreased by Cortical Photothrombosis

Stroke-induced functional impairments were assessed using multiple sensitive behavioral tasks. Locomotor activity was measured using the open field test. At baseline, all groups showed similar locomotor activity. The distance and velocity decreased over time after photothrombosis (P<0.01) (Figure 3A and 3B). ANOVA for repeated measures also showed a significant time × group interaction (P<0.05) indicating time-dependent differences in distance and speed among groups. Female mice were faster compared with males (P<0.05). Counterclockwise rotation decreased in all groups after photothrombosis (P<0.001; Table S1). Counterclockwise rotation was more robust in females (P<0.05).

Figure 3.

Figure 3. Behavioral outcome. Distance (A) and velocity (B) showed a decrease in overall activity and mobility in the open field test. Open field behavior was also used as a measure of anxiety. Mice spent most of the time near the wall, only occasionally visiting the center zone (C). Heatmap representation showed a lower activity during the follow-up (D). There were no differences among experimental groups at any time point. In the cylinder test, a significant time effect indicated persistent impairment (E). Grid walking test showed an increase in the number of forelimb foot-faults (F). Behavioral outcome was not different among experimental groups in selected tests. Values are mean±SD. n=11–19 per group.

Mice prefer to spend most of their time near the walls and avoid the open center23 and this is usually further exaggerated by cerebral ischemia. Here thigmotaxis (ie, time near wall) at baseline was lower in mice transplanted with human iPSC-derived astrocyte progenitors (Table S1). The time spent and frequencies of visits in the center of the open field were used as a measure of anxiety-like behavior. Indeed, mice spent most of the time near the wall, only occasionally visiting the center zone (Figure 3C and 3D; Table S1). Cumulative time spent in any zone was not affected by time, group, or sex.

Minor Cortical Lesion Produced a Long-Lasting Impairment in Sensorimotor Functions but Not in Gait

Exploratory activity and spontaneous use of impaired (contralateral) and nonimpaired (ipsilateral) forelimbs were measured by cylinder test (Figure 3E). At baseline, there were no differences between groups. There was a significant time effect (P<0.001) indicating persistent impairment without recovery. However, no overall group or sex effects or time interactions were observed.

Grid walking was used to assess motor coordination during locomotion. Photothrombosis induced a significant increase in number of foot-faults by impaired forelimb with slight spontaneous recovery during the follow-up (P<0.001; Figure 3F). There were no differences between groups or sex or interactions with time. Behavior of nonimpaired forelimbs was not affected by ischemia. Foot-faults by hindlimbs were minimal and were not counted.

CatWalk was used to characterize gait in mice. Males and females were tested separately by different detection settings to correct differences in body weight. Despite different settings, however, body weight correlated with many of the gait parameters (Table S2). Only a few scattered significant differences among groups were found at baseline (Table S2), indicating that cell transplantation itself did not affect gait. Repeated ANOVA showed a significant time effect, particularly in spatial parameters (eg, decrease in maximum contact area after photothrombosis) (Figure S3A; Table S3). Interestingly, there was a sex x time interaction in most of the spatial parameters for both left and right limbs. As an example, a closer examination revealed a transient decrease in maximum contact area followed by an increase only in males, which were both related to changes in body weight (Figure S3B).

Human iPSC-Derived Astrocyte Progenitors Engrafted in the Corpus Callosum

We next asked whether the genotype of the transplanted astrocyte progenitors affected their overall distribution in the ischemic brain. The specificity of GFAP antibodies allowed double fluorescent stainings (Figure S4). While the transplanted human cells were scattered relatively equally throughout the brain, human GFAP-positive astrocytes were located mainly in the corpus callosum (Figure 4A and 4B). We then measured staining intensities for ipsilateral and contralateral corpus callosum. hGFAP staining was not detected in mCRTL (Figure 5A). Ipsilateral values were higher in hAD (P<0.01) and hCTRL (P<0.05) groups. Sex did not affect the values, but interestingly, there was group × sex interaction (P<0.05) in both sides, possibly due to higher values in female mice.

Figure 4.

Figure 4. Overall distribution of host GFAP (glial fibrillary acidic protein) and hGFAP (human glial fibrillary acidic protein) stained astrocytes. Low magnification image of host GFAP and hGFAP stained astrocytes in the mouse with cortical photothrombosis (A). Distribution of human and host astrocytes in the corpus callosum (B). Scale bar=10 μm (B).

Figure 5.

Figure 5. Complex phenotype of human astrocytes in the corpus callosum. Integrated densities of human (A) and host (B) astrocytes were different in the corpus callosum. High magnification fluorescence images from the human astrocytes (C) and host astrocytes (E) in the corpus callosum. Human astrocytes had a more complex phenotype with longer processes compared with host astrocytes (D). Black symbols for males, red symbols (color online only) for females. Values are mean±SD. Scale bar=10 μm (C, E). hAD indicates human iPSC-astrocyte progenitors derived from AD patients; hCTRL, healthy donor control; and iCTRL, isogenic control astrocytes. *P<0.05; ***P<0.001; ***P<0.05 compared with mouse control (mCTRL) group; #P<0.05; ##P<0.01 compared with contralateral and ipsilateral side (A, B). **P<0.01 compared with mCTRL group (D).

The integrated density for host astrocytes in mCTRL was much higher compared with human astrocytes in the corpus callosum. There was also a difference between contralateral and ipsilateral sides (P<0.001; Figure 5B). Values for host astrocytes in the mCTRL group were lower compared with the other groups in the contralateral side (P<0.05; 0.001). No significant differences were found in the ipsilateral corpus callosum. Sex did not affect the staining intensity.

Overall, the phenotype of human astrocytes was more complex compared with host ones (Figure 5C and 5E; Table S4). To analyze the astrocyte phenotype in more detail, maximum intensity projections were generated from fluorescence images, converted to binary images, and skeletonized for the measurement of process lengths and end points (Figure S5). There was an overall group effect in the length of processes per cell (P<0.01) due to the increase in hAD and iCTRL groups (P<0.05; P<0.01; Figure 5D).

Host Astrocytes but Not Human Astrocytes Formed a Glial Scar

To better understand the behavioral results, we delineated a 100 µm-wide ROI at the border of the ischemic core in the cortex to measure glial responses to ischemia. Overall, integrated densities for host GFAP were much higher compared with hGFAP but were not different between the groups (Figure 6A through 6D). In the case of hGFAP, there was a significant overall group effect (P<0.001) due to lack of staining in mCTRL. mCTRL was different from hAD (P<0.05), iCTRL (P<0.01), and hCTRL (P<0.001; Figure 6C) groups. hGFAP staining intensities were higher in females (P<0.05).

Figure 6.

Figure 6. Different behavior of host and human GFAP (glial fibrillary acidic protein)-positive cells in the perilesional cortex. Integrated density for host GFAP (A) and hGFAP (human glial fibrillary acidic protein; C) positive cells was measured in the perilesional cortex after the behavioral follow-up. Staining for host GFAP showed a strong glial scar formation around the ischemic core (B). There were no differences in the integrated density among experimental groups. Only nonspecific staining for hGFAP was observed in mouse control group (mCTRL). Staining pattern for hGFAP was completely different and only a few scattered hGFAP-positive astrocytes were present in human iPSC-astrocyte progenitors derived from AD patients (hAD), isogenic control astrocytes (iCTRL), and healthy donor control (hCTRL) groups (D). White dashed line indicates lesion border. hGFAP staining was higher in females (red symbols [color online only]; P<0.05; A). Values are mean±SD. Scale bar=50 μm (B1–B4, D1–D4). *P<0.05; **P<0.01; ***P<0.001 compared with mCTRL group.

Aβ Deposits Were Not Present in PSEN1 ΔE9 Astrocyte-Transplanted Mice

To evaluate whether transplanted human cells produced Aβ deposits, brain sections were stained with rodent- and human-specific antibodies (Figure S6). As positive control, sections from APdE9 mice were used (Figure S6B and S6D). Aβ deposits were not seen in human control or PSEN1 ΔE9 astrocyte-transplanted mice. Adjacent to lesion core, rodent Aβ staining was present in all groups (Figure S6A).

Discussion

We generated chimeric mice to address for the first time how human astrocytes and astrocytic PSEN1 ΔE9- genetic background impacted the stroke severity and outcome. Infarct size or behavioral performance was not different among experimental groups, possibly due to minimal glial scar formation by human astrocytes.

A high number of HuNu-positive transplanted cells with variable phenotypes were widely distributed in the host brain after transplantation. Although not quantified, HuNu-positive cells co-stained with hGFAP, vimentin, PDGFRα, and DCX, suggesting generation of different glial cells, oligodendrocytes and neuronal progenitor phenotype. In line, Windrem et al20,24 showed that transplanted glial progenitors engraft and differentiate mainly into oligodendrocytes in the white matter in shiverer, myelin-deficient mice. In addition, majority of host astrocytes are replaced by their human counterparts in shiverer mice, which was not the case in our study.

Only a low number of hGFAP positive cells were found in the cortex. One should note, however, that in the healthy mouse brain GFAP content is low in cortex, subpopulations of resting astrocytes do not express the GFAP microfilament protein,25 and GFAP immunoreactivity decreases after trauma.26 The lack of GFAP expression does not necessarily prevent scar formation, as shown in GFAP/− mice.27 Interestingly, the majority of human GFAP-positive astrocytes remained engrafted in the corpus callosum, distant from the cortical lesion site. Moreover, the present data showed that transplanted cells with GFAP expression retain complex human astrocyte-specific morphology in the host ischemic environment, with processes being much longer in comparison to host astrocytes, in line with the previous study.5

Glial response to cortical photothrombosis is extensively studied in rodents.28–30 Astrocytes are important in limiting early excitotoxicity and forming a glial scar to separate the ischemic core from surrounding healthy tissue, a scar which later secretes proteoglycans inhibiting axonal growth and regeneration in perilesional tissue.31 We showed a strong scar formation by host astrocytes most likely due to astrocyte proliferation within the region adjacent to the lesion core. In contrast, the number of human astrocytes was small in the perilesional cortex and their participation in glial scar formation was not observed. The reason for this may be higher susceptibility of human astrocytes to oxidative stress leading to low survival rate.6 It may also be that human astrocytes were not activated by host ischemic signals and thus did not affect behavioral recovery as we hypothesized. Single-cell RNA sequencing is needed to study further host versus transplanted human astrocyte responses to ischemic insult.

Increasing evidence suggests that cerebrovascular diseases and AD not only coexist but interact, which leads to an exacerbated outcome in experimental animals12,14 and patients.11,32,33 Importantly, astrocytes are implicated in both disease pathologies.15 Of note, iPSCs derived from AD patients with pathogenic PSEN1 ΔE9 mutation are characterized by increased Aβ1-42 production and altered cytokine release (eg, IL-2, IL-6, IL-10) in vitro.16 To our surprise, the astrocyte AD pathology did not exacerbate ischemic damage or behavioral impairment in mice, possibly due to the above discussed low number of hGFAP-positive astrocytes in the perilesional tissue. In addition, we were not able to show Aβ deposits in PSEN1 ΔE9 astrocyte-transplanted mice indicating that pathology does not spread to host tissue.

Sex-related differences exist in many aspects of stroke from epidemiology to acute treatment and outcomes.34 The importance of using both sexes in experimental studies has been highlighted most recently by the STAIR consortium.35 Indeed, also in our study numerous sex-specific differences were present. It is known that reproductive hormones provide natural cerebrovascular protection in women during premenopausal years.36 After that, the rates of ischemic stroke begin to increase concomitant with the onset of menopause and loss of female sex hormones,37 and this might have contributed to the observed larger infarct volumes in females. However, although estrous cycle was not examined, mice at age of 14 months in the present study were expected to be reproductively senescent.38 In addition, there are sex-specific differences in ischemic cell death pathways, autophagy, and immune responses.39–41

Our study has several technical limitations. Only iPSCs from male donors and a limited number of lines were used to keep the study design feasible. Indeed, sex-specific cellular features may lead to different responses to ischemia.42 The observed sex-related differences in CatWalk were mainly due to body weight, which should be carefully considered in future studies.43 The follow-up time was rather long (14 months) and transplanted cells may have retracted to quiescent state or were differentiated to oligodentrocytes.44 Although GFAP is sensitive in detecting reactive astrocytes that respond to brain ischemia, a panel of additional astrocytic markers such S100b should have been included to assess glial phenotypes and responses more precisely.45 Last, immunodeficient nature of the mice may confound behavioral outcomes after stroke.

In conclusion, human glial progenitor cells were transplanted into neonatal Rag1 mice generating chimeric mice to recapitulate the human condition. The survival of transplanted cells was high and cells migrated widely in the host brain after cortical photothrombosis. However, only a minority had a GFAP-positive glial phenotype, formed glial scar, or impaired behavioral outcome. Caution is needed in using human glial chimeric mice in stroke research.

Article Information

Acknowledgments

N.-N. Välimäki carried out histological stainings; A. Bakreen, S. Häkli, and Y. Dunlop were responsible for behavioral testing and analysis; H. Dhungana operated mice; M.H. Keuters transplanted cells; M. Koskuvi, V. Keksa-Goldsteine, M. Oksanen, and H. Jäntti characterized and produced iPSCs and iPSC-derived astrocytes. Š. Lehtonen, T. Malm, J. Koistinaho, and J. Jolkkonen conceived the experiment, interpreted the results, and wrote the article.

Supplemental Material

Supplemental Materials and Methods

Figures S1–S6

Tables S1–S4

References 46–50

Nonstandard Abbreviations and Acronyms

beta-amyloid

AD

Alzheimer disease

AQP4

aquaporin4

GFAP

glial fibrillary acidic protein

hAD

human iPSC-astrocyte progenitors derived from AD patients

hCTRL

healthy donor control astrocytes

HuNu

human nuclei

iCTRL

isogenic control astrocytes

iPSC

induced pluripotent stem cell

mCTRL

mouse controls

PDGFRα

platelet-derived growth factor receptor alpha

PSEN1 ΔE9

PSEN1 exon 9 deletion

Disclosures None.

Footnotes

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.122.039700.

For Sources of Funding and Disclosures, see page 3200.

Correspondence to Jukka Jolkkonen, PhD, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. Email

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