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Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia.

ABSTRACT: Advancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of Aβ plaques, dystrophic neurites around plaques, synapse loss, dendrite retraction, axon fragmentation, phospho-Tau induction, and neuronal cell death in one model. We showed that the human iPSC microglia internalized and compacted Aβ to generate and surround the plaques, thereby conferring some neuroprotection. We investigated the mechanism of action of anti-Aβ antibodies protection and found that they protected neurons from these pathologies and were most effective before pTau induction. Taken together, these results suggest that this model can facilitate target discovery and drug development efforts.

SUBMITTER: Bassil R

PROVIDER: S-EPMC8410795 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia.

Bassil Reina R Shields Kenneth K Granger Kevin K Zein Ivan I Ng Shirley S Chih Ben B

Nature communications 20210901 1

Advancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of ...[more]

PMID: 34471104

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Project description:BackgroundWidescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells.MethodsiPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aβ42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aβ42.ResultsAD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100β and increased secretion and phagocytosis of Aβ42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia.ConclusionOur study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aβ42 production and phagocytosis.

Project description:BackgroundCellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia.ResultsAfter validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions.ConclusionsNeurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

Dataset Information

Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia.

Publications

Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia.

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