iPSC-derived Astrocytes Research Articles

Establishment of autaptic culture with human-induced pluripotent stem cell-derived astrocytes

SummaryAlthough astrocytes are involved in the pathogenesis of CNS diseases, how they induce synaptic abnormalities is unclear. Currently, in vitro pathological astrocyte cultures or animal models do not reproduce human disease phenotypes accurately. Induced pluripotent stem cells (iPSCs) are replacing animal models in pathological studies. We developed an autaptic culture (AC) system containing single neuron cultures grown on microislands of astrocytes. AC with human iPSC-derived astrocytes (HiA) was established. We evaluated the effect of astrocytes on the synaptic functions of human-derived neurons. We found a significantly higher Na+ current amplitude, membrane capacitance, and number of synapses, as well as longer dendrites, in HiAACs compared with neuron monocultures. Furthermore, HiAs were involved in the formation and maturation of functional synapses that exhibited excitatory postsynaptic currents. This system can facilitate the study of CNS diseases and advance the development of drugs targeting glial cells.

Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans

ObjectiveMutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. MethodsWe conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. ResultsWe identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5–7 Hz spike-wave discharges and absence seizures. InterpretationSLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening

Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.Graphical abstract

MS-Driven Metabolic Alterations Are Recapitulated in iPSC-Derived Astrocytes.

ObjectiveAstrocytes play a significant role in the pathology of multiple sclerosis (MS). Nevertheless, for ethical reasons, most studies in these cells were performed using the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism.MethodsWe obtained and characterized induced pluripotent stem cell (iPSC)‐derived astrocytes from three PwMS and three unaffected controls, and performed electron microscopy, flow cytometry, cytokine and glutamate measurements, gene expression, in situ respiration, and metabolomics. We validated our findings using a single‐nuclei RNA sequencing dataset.ResultsWe detected several differences in MS astrocytes including: (i) enrichment of genes associated with neurodegeneration, (ii) increased mitochondrial fission, (iii) increased production of superoxide and MS‐related proinflammatory chemokines, (iv) impaired uptake and enhanced release of glutamate, (v) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (vi) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS.InterpretationHere we describe the metabolic profile of iPSC‐derived astrocytes from PwMS and validate this model as a very powerful tool to study disease mechanisms and to perform non‐invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies. ANN NEUROL 2022;91:652–669

Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons

Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our invitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.

Insights into Human-Induced Pluripotent Stem Cell-Derived Astrocytes in Neurodegenerative Disorders.

Most neurodegenerative disorders have complex and still unresolved pathology characterized by progressive neuronal damage and death. Astrocytes, the most-abundant non-neuronal cell population in the central nervous system, play a vital role in these processes. They are involved in various functions in the brain, such as the regulation of synapse formation, neuroinflammation, and lactate and glutamate levels. The development of human-induced pluripotent stem cells (iPSCs) reformed the research in neurodegenerative disorders allowing for the generation of disease-relevant neuronal and non-neuronal cell types that can help in disease modeling, drug screening, and, possibly, cell transplantation strategies. In the last 14 years, the differentiation of human iPSCs into astrocytes allowed for the opportunity to explore the contribution of astrocytes to neurodegenerative diseases. This review discusses the development protocols and applications of human iPSC-derived astrocytes in the most common neurodegenerative conditions.

Multielectrode Arrays for Functional Phenotyping of Neurons from Induced Pluripotent Stem Cell Models of Neurodevelopmental Disorders.

Simple SummaryMultielectrode array technology allows researchers to record the spontaneous firing activity of cultured neurons over a period of multiple weeks or months. These data can be valuable for understanding how the functional relationships between neurons evolve as they begin to form connections and wire into a functional network. This technology has been adopted by researchers using stem cells to produce human neurons in culture to study neurodevelopmental disorders. However, the dizzying complexity and scale of the data generated have posed some challenges with the analysis and interpretation of experimental results. Here, we first provide historical context as to why multielectrode array platforms were originally developed, and use this perspective to explore some of the challenges currently facing the field. We then highlight new analysis methods, provide some guidance for improving the analysis of multielectrode array data, and discuss standardizing how these findings are communicated in scientific publications.In vitro multielectrode array (MEA) systems are increasingly used as higher-throughput platforms for functional phenotyping studies of neurons in induced pluripotent stem cell (iPSC) disease models. While MEA systems generate large amounts of spatiotemporal activity data from networks of iPSC-derived neurons, the downstream analysis and interpretation of such high-dimensional data often pose a significant challenge to researchers. In this review, we examine how MEA technology is currently deployed in iPSC modeling studies of neurodevelopmental disorders. We first highlight the strengths of in vitro MEA technology by reviewing the history of its development and the original scientific questions MEAs were intended to answer. Methods of generating patient iPSC-derived neurons and astrocytes for MEA co-cultures are summarized. We then discuss challenges associated with MEA data analysis in a disease modeling context, and present novel computational methods used to better interpret network phenotyping data. We end by suggesting best practices for presenting MEA data in research publications, and propose that the creation of a public MEA data repository to enable collaborative data sharing would be of great benefit to the iPSC disease modeling community.

Rapid Generation of Ventral Spinal Cord-like Astrocytes from Human iPSCs for Modeling Non-Cell Autonomous Mechanisms of Lower Motor Neuron Disease.

Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human-induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. Here, we describe the efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord. These induced astrocytes exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal cord features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS.

Astrocytic 4R tau expression drives astrocyte reactivity and dysfunction.

The protein tau and its isoforms are associated with several neurodegenerative diseases, many of which are characterized by greater deposition of the 4-repeat (4R) tau isoform; however, the role of 4R tau in disease pathogenesis remains unclear. We created antisense oligonucleotides (ASOs) that alter the ratio of 3R to 4R tau to investigate the role of specific tau isoforms in disease. Preferential expression of 4R tau in human tau–expressing (hTau-expressing) mice was previously shown to increase seizure severity and phosphorylated tau deposition without neuronal or synaptic loss. In this study, we observed strong colocalization of 4R tau within reactive astrocytes and increased expression of pan-reactive and neurotoxic genes following 3R to 4R tau splicing ASO treatment in hTau mice. Increasing 4R tau levels in primary astrocytes provoked a similar response, including a neurotoxic genetic profile and diminished homeostatic function, which was replicated in human induced pluripotent stem cell–derived (iPSC-derived) astrocytes harboring a mutation that exhibits greater 4R tau. Healthy neurons cultured with 4R tau–expressing human iPSC–derived astrocytes exhibited a higher firing frequency and hypersynchrony, which could be prevented by lowering tau expression. These findings support a potentially novel pathway by which astrocytic 4R tau mediates reactivity and dysfunction and suggest that astrocyte-targeted therapeutics against 4R tau may mitigate neurodegenerative disease progression.

A Three-Dimensional Brain-on-a-Chip Using Human iPSC-Derived GABAergic Neurons and Astrocytes.

Brain-on-a-chip is a miniaturized engineering platform to mimic the structural and functional aspects of brain tissue. We describe a method to construct a three-dimensional (3D) brain-on-a-chip in this chapter. We firstly portray the method of a brain-on-a-chip model with cocultured mice neurons, microglia, and astrocytes to mimic brain tissue and membrane-free perfusion with endothelial cells, in which we successfully build the blood-brain barrier to screen neurotoxicity. Then we describe a method to construct a brain-on-a-chip with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded GABAergic neurons and astrocytes and a perfusion channel with dynamic flow. We also include the broader applicability test of this model using an organophosphate (OP), malathion, to induce acute and chronic neurotoxicity, and then using butyrylcholinesterase (BuChE) as an exogenous bioscavenger of OP. Following the methods listed in this chapter, we are able to measure the neurotoxic effects on construct integrity, viability, and total AChE and BuChE activity.

Construction of minimal neural circuits by iPSC-derived astrocytes for glial drug discovery

Construction of minimal neural circuits by iPSC-derived astrocytes for glial drug discovery

Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies.

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions.

Compound screen identifies the small molecule Q34 as an inhibitor of SARS-CoV-2 infection

SummaryThe COVID-19 outbreak poses a serious threat to global public health. Effective countermeasures and approved therapeutics are desperately needed. In this study, we screened a small molecule library containing the NCI-DTP compounds to identify molecules that can prevent SARS-CoV-2 cellular entry. By applying a luciferase assay-based screening using a pseudotyped SARS-CoV-2-mediated cell entry assay, we identified a small molecule compound Q34 that can efficiently block cellular entry of the pseudotyped SARS-CoV-2 into human ACE2-expressing HEK293T cells, and inhibit the infection of the authentic SARS-CoV-2 in human ACE2-expressing HEK293T cells, human iPSC-derived neurons and astrocytes, and human lung Calu-3 cells. Importantly, the safety profile of the compound is favorable. There is no obvious toxicity observed in uninfected cells treated with the compound. Thus, this compound holds great potential as both prophylactics and therapeutics for COVID-19 and future pandemics by blocking the entry of SARS-CoV-2 and related viruses into human cells.

APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes.

SummaryThe apolipoprotein E4 (APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs) (APOE4, E3, E2, and APOE knockout [APOE-KO]) to functional “iAstrocytes”. Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 > E3 > E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 > E3 > E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 > E3 > E2 > KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.

Functional exploration of AGFG2, a novel player in the pathology of Alzheimer disease.

There are three main clinical presentations in Alzheimer disease (AD) with diversity in phenotype, onset and progression of clinical symptoms: autosomal dominant (ADAD), early onset (EOAD) and late onset (LOAD). Ultimately, AD is characterized by the deposition of Aβ and ptau protein aggregates in the brain. This work aims to identify and characterize common disrupted pathways across AD etiologies. We examined bulk transcriptomic data from brain donors to the DIAN (ADAD, N=19) and Knight-ADRC (EOAD, N=13; LOAD, N=55; controls, N=16). We performed differential gene expression (DGE) analyses using DSeq2 and cross-checked significant signals with known GWAs loci. We replicated our findings in three AMP-AD independent studies. DGE analysis identified 57 significantly differentiated genes across ADAD, EOAD or LOAD vs controls. Among those, AGFG2 (p=7×10-4 ) had a higher expression in cases and falls under the AD GWAs signal for NYAP1 (rs1476679). This effect replicated in all independent datasets: Mount Sinai (p=8.63×10-3 ), Mayo (p=5.88×10-12 ), ROSMAP (p=3.96×10-05 ). AGFG2 is expressed primarily in astrocytes and is a member of the HIV-1 Rev binding protein family that mediates the nucleocytoplasmic transfer of proteins and RNAs. AGFG2 has been implicated in APP metabolism, leading to the hypothesis that higher expression of AGFG2 could promote more release of APP to the media. Our results suggest that AGFG2 may be a novel player in the etiology of AD. We are currently modifying AGFG2 expression on iPSC-derived astrocytes using CRIPR-Cas9 technology to evaluate AGFG2 role in APP and Tau metabolism. We will present these results at the conference.

Investigating astrocytes as mediators of tau spread.

Highly phosphorylated tau aggregates spread in a prion-like manner through tauopathy brain. Increasing evidence suggests that astrocytes may influence tau spread, with recent data describing mechanisms of tau uptake my astrocytes. However, the efficiency of uptake of different disease-associated tau species is not well defined, nor is the effects of tau uptake on astrocyte reactivity and function. We have investigated the relationship between tau seeds and reactive astrocytes in htau mice and in human astrocyte cultures. Tau was isolated from human postmortem control (CTRL), Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and Pick's disease (PiD) brain and injected intraperitoneally into 4-month old wild-type and htau mice. Brains and other tissues were collected for biochemical and immunohistochemical analysis of tau aggregated burden in different brain regions when mice reached 18 months of age. Human iPSC-derived astrocytes and iNPC-astrocytes were established and their gene and protein expression profiles characterised. Aggregated tau was isolated from postmortem human brain for spiking into astrocyte cultures. Cells were analysed for efficiency of tau uptake and astrocyte reactivity. Peripheral injection of brain extracts from AD, PSP and PiD brain resulted in regional differences in the patterns of tau aggregate accumulation in htau mouse brain. Tau pathology was commonly associated with reactive astrocytes. Cultures of human iPSC-derived astrocytes and iNPC-astrocytes that express mature astrocyte markers were established. Both types of cell culture were found to internalise tau extracts from human postmortem brain. Furthermore, preliminary analysis indicates alterations in astrocyte reactivity following tau uptake. Our data suggests that neurons in different regions are selectively vulnerable to tau aggregates from different tauopathies. Our human astrocyte cultures demonstrate that astrocytes can internalise tau seeds and alter their reactivity and potentially their functions. Further investigations will determine the mechanisms of tau uptake and the downstream effects on astrocyte signalling and functional changes, providing further insights into how astrocytes may affect the spread of tau and potentially contribute to selective regional vulnerability in tauopathies.

IPCS-derived APPswe mutant pericytes produce beta-amyloid and induce the activation of co-cultured astrocytes.

The amyloid cascade hypothesis has been the most prevalent theory so far to explain the molecular mechanisms underlying the pathogenesis and progression of Alzheimer's disease (AD). In parallel, substantial evidence indicates contribution of cerebrovascular dysfunction to development of AD in the early stage of the disease. Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder characterized by beta-amyloid (Ab) accumulation in the walls of small and medium vessels in the brain and meninges. Nearly 90 percent of AD patients show CAA pathology linked to vascular dysfunction, eventually leading to hemorrhage lesions in the brain. Pericytes in the blood-brain barrier (BBB) are crucial for stabilizing the vasculature structure. Substantial pericyte loss has been reported in AD, which correlates with the BBB breakdown and decline of cognitive functions. However, the exact role of pericytes in the AD-associated vascular dysfunction and AD progression remain unclear. To investigate whether AD pericytes contribute to BBB impairment, we differentiated pericytes from induced pluripotent stem cell (iPSC) lines generated from two APPswe mutant and two healthy control individuals. These generated pericytes were characterized with qPCR or immunostaining to ensure the expression of classic pericyte markers, such as PDGFRb, a-SMA, and LAMA2. Then, pericytes were co-cultured with control iPSC-derived astrocytes to test the interaction of pericytes with another key cell type in BBB. We found that the GFAP immunoreactivity was increased in those astrocytes co-cultured with APPswe mutant pericytes, indicating activation of astrocytes. We hypothesized that astrocyte activation is triggered by Aβ deposition. Hence, we collected medium from pericyte monocultures after seven days of cultivation and detected by ELISA approx. 64.8 (±23.5) pg/ml of Aβ42 secreted by APPswe pericytes. In control cultures Aβ42 remained undetectable. These results indicate that APPswe pericytes are capable of producing toxic amounts of Aβ42, which could potentially contribute to CAA and affect other surrounding cell types. This is a novel concept since CAA is believed to be caused primarily by neuron-derived Aβ42.

Human iPSC-derived astrocytes transplanted into the mouse brain undergo morphological changes in response to amyloid-\u03b2 plaques

BackgroundIncreasing evidence for a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer’s disease comes from molecular and functional studies in rodent models. However, these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression.ResultsTo address these challenges, we established an approach to study human astrocytes within the mouse brain by transplanting human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors into neonatal brains. Xenografted hiPSC-derived astrocyte progenitors differentiated into astrocytes that integrated functionally within the mouse host brain and matured in a cell-autonomous way retaining human-specific morphologies, unique features, and physiological properties. In Alzheimer´s chimeric brains, transplanted hiPSC-derived astrocytes responded to the presence of amyloid plaques undergoing morphological changes that seemed independent of the APOE allelic background.ConclusionsIn sum, we describe here a promising approach that consist of transplanting patient-derived and genetically modified astrocytes into the mouse brain to study human astrocyte pathophysiology in the context of Alzheimer´s disease.