Generation of S1p fraction: Frozen hippocampus and cortical tissues of 9-month old PS19 mice were weighed (100mg-250mg) and put in Beckman Centrifuge Tube, polycarbonate thick wall (cat # 362305). A 10 × volume of homogenization buffer was used to homogenize brain tissue with Hsaio TBS buffer (50 mM Tris, pH 8.0, 274 mM NaCl, 5 mM KCl) supplemented with protease and phosphatase inhibitor cocktails (Roche, cat#05892791001 and cat#04906837001), as described previously3,23. Briefly, the homogenate was centrifuged at 48,300 g for 20 min at 4 °C. The supernatant was then centrifuged a second time at 186,340 g at 4°C for 40 min. The TBS-extractable pellet (S1p) fraction was resuspended in a 4x volume of TE buffer relative to the starting weight of the tissue homogenate, aliquoted and frozen at -80°C.
S1p fraction quantification:
Immuno-depletion of tau from S1p fraction: Tau aggregates in S1p fractions were eliminated from the fractions by a direct immuno-precipitation kit (Pierce, cat# 26148). Briefly, first tau-5 antibody was coupled to AminoLink plus Coupling Resin, and the fractions were pre-cleared using the Control Agarose Resin with all the materials provided by the kit. The sample was added to the antibody-coupled resin in the spin column and incubated in the column for overnight at 4°C on a gentle rotator. The column was centrifuged, and the flow-through saved for further experimentation. After 3 washes with IP buffer, the spin column was placed into a new collection tube, and tau plus antibodies were eluted from the resin. The eluate was analyzed for presence of tau.
Cell Culture and Treatment
All cell cultures were maintained at 37 °C with 5% CO2. All cell counts were performed in quadruplicate using the Cellometer K2 with AOPI viability dye (Nexcelom).
Neural Progenitor Cell (NPC) Culture: Human iPSC (XCL-1) derived neural progenitor cells (NPCs, Stem Cell Tech 70901) were maintained in serum-free STEMdiff™ Neural Progenitor Medium 2 (Stem Cell Tech 08560 ) on Corning® Matrigel® hESC-qualified Matrix (Corning 354277) coated tissue culture plates. NPCs were plated at 50,000 cells/cm2 and passaged at 90% confluency by AccutaseTM (Stem Cell Tech 07920) dissociation as necessary. A full media change was performed every other day. Low passage (passage <3) NPCs were cryopreserved in STEMdiff™ Neural Progenitor Medium 2 with 10% DMSO, and all NPCs used for experimentation were maintained at passage <6.
iPSC Neuronal Cell (hiNC) Differentiation: NPCs were passaged and plated at 50,000 cells/cm2 in STEMdiff™ Forebrain Neuron Differentiation Media (Stem Cell Tech 08600) on Corning® Matrigel® coated tissue culture treated plates and transduced with a NEUROG2 lentivirus (Genecopia LPP-T7381-Lv105-A00-S) at MOI 3 to induce iPSC derived neuronal cells (hiNC). After 24 hours of transduction, a full media change was performed.
iPSC Astrocytic Cell (hiAC) Differentiation: iPSC derived astrocytic cells (hiAC) were differentiated from NPCs by small molecule differentiation in STEMdiff™ Astrocyte Differentiation Media (Stem Cell Tech 100-0013) on Corning® Matrigel® coated tissue culture treated plates. A full media change was performed daily for four days, and cultures were passaged at 90% confluence by AccutaseTM. Cells were reseeded at 150,000 cell/cm2 and culture was continued in STEMdiff™ Astrocyte Differentiation Media with a full media change every other day for 14 days, passaging as necessary with AccutaseTM. At this stage the differentiated Astrocyte Precursor Cells (APCs) were cryopreserved in STEMdiff™ Astrocyte Differentiation Media with 10% DMSO. At time of use APCs were thawed and plated at 150,000 cell/cm2 in STEMdiff™ Astrocyte Maturation Media (Stem Cell Tech 100-0016) on Corning® Matrigel® coated tissue culture treated plate. A full media change was performed every other day for 6 days, with one passage by AccutaseTM at 90% confluence as necessary.
Asteroid Generation and Maintenance: A single cell suspension of hiNCs and hiACs was prepared by AccutaseTM dissociation and washed once with DMEM/F12 (Stem Cell Tech 36254) to remove debris. hiNCs and hiACs were combined at a 1:1 ratio in Asteroid Media (DMEM/F12 (Stem Cell Tech 36254) , 1% Glutamax (Thermo Scientific 35050061), 1% Sodium Pyruvate (Thermo Scientific 11360070), 1% N-2 Supplement (Thermo Scientific 17502-048), 1% B-27 Supplement (Thermo Scientific 17504044), 10 uM Y-27632 (EMD Millipore SCM075) 1% PenStrep (Thermo Scientific 15140148), 1 mg/mL Heparin (Sigma-Aldrich H3149-250KU)) and plated in AggreWellTM800 microwells (Stem Cell Tech 34815) coated with Anti-Adherence Rinsing Solution(Stem Cell Tech 07010). The AggreWell plate was immediately centrifuged at 100xg for 3 minutes to capture the cells in the microwells and incubated for 24 hours. A half media change was performed at 24 hours and then every other day for one week. At one week when the spheroids displayed a smooth, bright edge under the cell culture microscope cultures were transferred to ultra-low attachment round bottom 96 well plates (Fisher Scientific 07-201-680) and maintained in 100-200 uL asteroid media rotating at 85 rpm. A half media change was performed every other day for up to three weeks.
hiNC oTau Treatment: hiNCs were selectively exposed to 0.04 mg/mL oTau by direct administration in cell culture media for 24 hours before incorporation into asteroid culture.
PU-H71 Treatment: Asteroids were treated with 1 uM PU-H71 by direct administration in cell culture media for 72 hours before timepoint collection.
Asteroid Fixation: At time of collection asteroids were transferred to a 1.5 mL Protein LoBind Eppendorf (Eppendorf 022-43-108-1) and allowed to settle. The supernatant was discarded and asteroids fixed in 4 °C 4% PFA in 1X PBS for 15 minutes, rotating at room temperature. After fixation, asteroids were washed 3x for 5 minutes each with 4°C 1X PBS, rotating at room temperature. Samples were stored in 1X PBS at 4 °C.
Conditioned Media Collection: 50 uL replicates of conditioned cell culture media from replicate asteroids were collected in flat bottomed 96 well plates and frozen at -20 °C.
Immuno-fluorescence labeling: For immuno-labeling, selected asteroids from each condition were washed in 150µl PBS for 10 mins in the U-bottom 96-well plate and then permeabilized in 150µl PBS/0.01% Triton X-100 (PBST). The asteroids were then blocked in PBST supplemented with 5% BSA and 5% normal donkey serum for 1.5-2 hrs at room temperature (RT). After blocking, asteroids were incubated in primary antibodies dilute in 5% BSA/PBST and for overnight at 4°C. On the second day, the asteroids were washed 3 times in PBST, 15 min each before they were transferring into 2° antibodies dilute (1:700 of Dylight-/Alexa-conjugated antibodies made in donkey purchased from Thermo Fisher Scientific in 5% BSA/PBST) for 2 hrs at RT. For DAPI nuclei stain, DAPI (1:10,000) was diluted in PBST and incubated with asteroids for 15 min followed by being washed 2x with PBST then 1x with PBS, 10 min each. The asteroids were then mounted onto microscope glass slides in Prolong gold antifade reagent. Primary antibodies used for asteroid labeling were as follows: Tuj1/beta3-Tubulin (chicken, SYSY, cat# 302 306, 1: 300), MAP-2 (rabbit, Millipore, cat# AB5622, 1: 1000), Rabbit monoclonal anti-S100β (Abcam, Cat# ab52642, 1:400); GFAP Monoclonal Antibody (Thermo Fisher Scientific, Cat#13-0300, 1:400); Mouse monoclonal anti-TOMA2 (provided by Dr. Rakez Kayed), 1:300; MC1 (provided by Dr. Peter Davies, Northwell), 1:300; CP-13 (provided by Peter Davies, Northwell), 1:300; AT-8 (provided by Dr. Nicholas Kanaan, Michigan State University), 1:300.
Thioflavin S staining: The fresh made Thioflavin S (ThioS) solution was prepared by dissolving 1g of ThioS (Millipore Sigma, Cat# T1892) in 100ml 80% ethanol and was kept stirring for overnight at 4 °C before filtered for final use. The asteroids to be stained were washed sequentially in 70% and 80% ethanol, 1 min each, prior to incubating in ThioS/80% ethanol solution for 15 min. Asteroids were then sequentially washed in 80% and 70% ethanol, 1 min each, followed by two rinses in PBS. Asteroids were mounted in Prolong Gold antifade reagent and stored in the dark until imaging.
Flouro Jade B staining: The Flouro jade B reagent was purchased from EMD Millipore (Cat# AG310-30MG) and the staining protocol was followed as instructed by the manufacture. Briefly, the staining solution was prepared from a 0.01% stock solution for Fluoro-Jade B that was made by adding 10 mg of the dye powder to 100 mL of distilled water. To make up 100 mL of staining solution, 4 mL of the stock solution was added to 96 mL of 0.1% acetic acid vehicle. This results in a final dye concentration of 0.0004%. The stock solution, when stored in the refrigerator was stable for months, whereas the staining solution was typically prepared within 10 minutes of use and was not reused. Before staining, the asteroids were rinsed in distilled water and were then treated with 0.06% KMnO4 solution for 15 min. Then the asteroids were stained with FluoroJade B working solution for 30 min followed by being washed with PBS 5 min twice. Asteroids were mounted in Prolong Gold antifade reagent and stored in the dark until imaging.
LDH Cytotoxicity Assay: The CytoTox 96 Non-Radioactive Cytotoxicity Assay was performed as per manufacturer’s instructions using 50 uL conditioned media replicates to measure lactate dehydrogenase (LDH) release (Promega G1780). Absorbance readings at 490 nm were taken on a SpectraMaxM5plate reader with SoftMax Pro 7.1 software.
Single Cell RNA Sequencing Sample Preparation and Sequencing: 30 asteroid per condition were pooled in a 1.5 mL Protein LoBind Eppendorf (Eppendorf 022-43-108-1) and allowed to settle. The supernatant was carefully discarded and a single cell suspension was produced by incubation in 500 uL digestion buffer (AccutaseTM with 80 U/mL Protector RNase Inhibitor (Sigma-Aldrich 03335402001)) for 1 hour at 37 °C with gentle pipette mixing every 10 minutes. At the end of the incubation the single cell suspension was washed with 500 uL wash buffer (0.02% BSA in 1X PBS with 80 U/mL Protector RNase Inhibitor) and passed through a 20 uM filter (MACS, Miltenyi Biotec 130-101-812) to a fresh 2 mL Protein LoBind Eppendorf. An additional 1 mL of wash buffer was then passed through the same filter for a total single cell suspension of 2 mL. The samples were centrifuged at 300 g for 5 minutes at 4 °C followed by another was in 1 mL wash buffer. After another centrifugation the supernatant was discarded and the single cell pellet gently resuspended in 50 uL wash buffer. Cells were counted in quadruplicate on the Cellometer K2 with AOPI and processed through the single cell RNA-sequencing pipeline from 10X Genomics, 3’ Version 3 (10X Genomic Chromium).
Briefly, the single cell suspension was mixed with RT reaction mix to target a 8000 cell recovery and 75 ul was loaded onto a chromium microfluidics chip with 40 μL of barcoded beads and 280 μL of partitioning oil. The chip was run on the chromium controller, encapsulating a single cell and barcoded bead within individual oil droplets. Reverse transcription was performed within these individual oil droplets to produce barcoded cDNA. cDNA was then isolated by Silane DynaBeads (Thermo Fisher Scientific, Dynabeads MyONE Silane, Cat# 37002D) before PCR amplification. Amplified cDNA cleanup and size selection was performed using SPRIselect beads (Beckman-Coulter, SPRIselect, Cat# B23317) and cDNA quality was assessed by the High-Sensitivity DNA assay (on the Agilent 2100 BioAnalyzer (Agilent, High-Sensitivity DNA Kit, Cat# 5067-4626). Sequencing libraries were then prepared according to 10X specification, including fragmentation, sequencing adaptor ligation, and sample index PCR. Between each of these steps, library cleanup and size selection was performed by SPRIselect beads. Final cDNA library quality was assessed by the Agilent BioAnalyzer High-Sensitivity DNA assay and the Qubit High-Sensitivity DNA assay and quality-confirmed libraries were sequenced on Illumina’s NextSeq 500 platform to a depth of 200 million paired-end reads.
Images Analysis: Images were captured by Carl Zeiss confocal LSM700. The immuno-fluorescence stained DAPI-positive cells in each image of asteroids were quantified by Image J with function of automatically cell counting. The staining intensity in immuno-fluorescence labeled asteroids were measured by ImageJ. The intensity of MC1, TOMA2, CP13, AT8, ThioS and Fluoro-jade B were normalized by DAPI numbers. Schematics were created with BioRender.com.
GraphPad Prism Statistical analysis :Statistical analyses and figures artwork were performed using GraphPad Prism version 9.00 for Windows with two sided α of 0.05. All group data are expressed as mean ± SEM. Colum means were compared using one-way ANOVA with treatment as the independent variable. And group means were compared using two-way ANOVA with factors on oTau treatment and time points, respectively. When ANOVA showed a significant difference, pair wise comparisons between group means were examined by Tukey’s, Dunnett or uncorrected Fisher’s LSD multiple comparison test. Significance was defined when p< 0.05. LDH assay data analysis was performed in with a paired t-test.
Single Cell RNA Sequencing Data Analysis
CellRanger Pipeline: CellRanger version 3.1.0 (10X Genomics) was used to combine and process the raw Illumina NextSeq 500 RNA sequencing data. First each sequencing library was demultiplexed by sample index to generate FASTQ files for paired-end reads using the CellRanger mkfastq pipeline. FASTQ files were then passed to the CellRanger count pipeline, which used STAR aligner32 to align reads to the human reference genome (GRCh38). The CellRanger aggr pipeline was then used to equalize the aligned molecule_info.h5 sample libraries across sequencing depths (by each sample cell being down-sampled to have the same confidently mapped reads per cell) and aggregated together to generate the gene-cell barcode matrix.
Seurat Object Filtration: Subsequent filtering, normalization, and scaling of data was performed using Seurat version 3.2.2 33,34. The Seurat object was created with a min.cells of 3 and a min.features of 200. Cells with less than 200 and greater than 5000 detected genes or greater than 20% mitochondrial counts were filtered out. Samples were subset to a max.cells.per.ident of 4642. Gene counts for each cell were normalized by total expression, multiplied by a scale factor of 10,000 and transformed to log scale. PCA based on the highly variable genes detected (dispersion of 2) was performed for dimension reduction and the top 20 principal components (PCs) were selected. We clustered cells based on graph-based methods (KNN and Louvain community detection method) implemented in Seurat. Clusters were visualized using uniform manifold approximation and projection (UMAP)35 .
Cluster Cell Type Identification: To identify neuronal cell type subpopulations, we performed differential expression analysis using the Wilcoxon rank-sum test implemented in Seurat between previously defined clusters with a min.pct or 0.1, logfc.threshold of 0.25, and a pseudocount of 1E4 . This identified top expressing genes for each cluster, which were then considered alongside the feature expression of canonical gene cell type markers to conclude cell type cluster identification.
DE analysis: Differential expression analysis was performed for each cell type between control and AstAD samples using the Wilcoxon rank-sum test implemented in Seurat with a min.pct of 0.1, a logfc.threshold of 0.1, and a pseudocount of 1E4. A multiple comparison correction was performed using the Benjamin & Hochberg FDR method to produce an adjusted p-value 36. Differentially expressed genes were evaluated according to their log fold change (greater than log2(.25)) and adjusted p values (0.05). All figures were generated using the ggplot2 R package and associated EnhancedVolcano R package 37,38.
Functional enrichment analysis: Functional geneset enrichment analysis of the significant differential genes between control and AstAD samples was performed using the R implemented GProfiler2 39. The enrichment analysis was run as an ordered query (ordered by log2FC) using a threshold of 0.05 and using Benjamin & Hochberg FDR for multiple testing correction36. Only genes in the Seurat dataset were considered by using a custom domain scope. A custom source GMT, gp__zSEF_sD9Q_d1M, was used. It includes all Hallmark gene sets, curated gene sets, and ontology gene sets from the Molecular Signatures Database (MsigDB) v7.2 40,41. The enrichment analysis was assessed and visualized by a heatmap of significance (−log10(p value)) of the top 20 enriched pathways per sample comparison. Comparative functional geneset enrichment analysis between AstAD and published datasets was performed with the same gprofiler2 settings, but as a non-ordered query. Gene input was as follows: From AstAD dataset, genes upregulated in NEU_A, NEU_B, and ASC at 21 DIV3D (n=129, p <0.05, log fold change > 0.25). From Grubman et al. 202025 genes upregulated in AD DEG 5 and 7 (n = 109, log fold change > 1 and FDR < 0.01).From Mathys et al. 202028 , genes upregulated in late-pathology cases that are common to ≥5 cell types (n=16, log fold change > 1 and FDR < 0.01)). The enrichment analysis was assessed and visualized by a heatmap of significance (−log10(p value)) of the top 10 enriched pathways per data set. All heatmaps were generated using the ComplexHeatmap R package and color scale generated using dependent R package circilize42. Additional visualization of significant (p< 0.01) enriched pathways was performed using Cytoscape EnrichmentMap43,44 with an edge cutoff of 0.375. Gene sets in EnrichmentMap cluster by similarity, and annotates of shared gene set features were added manually using Cytoscape implemented AutoAnnotate.
Module analysis: The scaled expression per cell of literature curated and MsigDB ontology genesets (see Source Table 1. Genesets) was compared between control and AstAD samples by computing the mean expression using colMeans and performing a t.test across comparison pairs using stat_compare_means. Figures were generated using the ggplot2 R package37.
Data Availability: Raw and processed scRNA-seq data are available from GEO under accession GSE165587. Processed scRNA-seq datasets are available on Single Cell Portal, including the cell barcodes, UMAP coordinates, and other available characteristics. The processed scRNA-seq data is available at https://singlecell.broadinstitute.org/single_cell/study/SCP1271/asteroid1. The source data underlying Fig. 2, 4 and Suppl. Fig. 4C, 7-10 are provided in the Source Data files as follows: Source data of differential gene expression and celltype_markers is available in supplemental file asteroid1_sourcefile_deg and source data of gprofiler2 functional geneset enrichment, Source Table 1, Source Table 2, and Source Table 3 are available in supplemental file asteroid1_sourcefile_gpro.
Code Availability: The original R scripts for Seurat processing are available on github [https://github.com/satijalab/seurat). All custom code to reproduce the analyses and figures reported in this paper are available on github (https://github.com/ChristineLab/asteroid1).