hPSC culture

The hESC line (SNUhES31) was obtained from the Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University Hospital, South Korea. The hiPSC lines from a patient with early-onset AD (56 years old, female) were kindly provided by Dr. Na-Yeon Jung (Department of Neurology, Pusan National University Yangsan Hospital, Pusan National University School of Medicine, Republic of Korea). The cells were cultured on 10 µg/mL mitomycin C-treated STO cells (ATCC) in hPSC medium (1% MEM-NEAA (Thermo Fisher Scientific), 1% GlutaMAX™ (Thermo Fisher Scientific), 7 µL/L β-mercaptoethanol, and 20% knockout serum replacement in DMEM/F-12) containing 20 ng/mL of basic fibroblast growth factor (bFGF). For feeder-free culture, the cells were cultured in Essential-8 medium (Thermo Fisher Scientific) on GeltrexTM-coated (Thermo Fisher Scientific) culture plates. The hPSCs were subcultured every four days using 500 µM EDTA-PBS.

Establishment of AD hPSC lines

We constructed the lentiviral plasmid vectors CAG-MCS, PGK-MCS, EF1α-MCS, and CMV-MCS by editing the pCAG-CreERT2, pSico-PGK-Puro, pCDH-EF1α-MCS-IRES-RFP (System Bioscience), and pLV-mCherry plasmids. pCAG-CreERT2 was gifted by Connie Cepko (Addgene, #14,797). The pSico-PGK-puro plasmid was gifted by Tyler Jacks (Addgene, #17,797), and pLV-mCherry was gifted by Pantelis Tsoulfas (Addgene, #36,084). Next, we generated CAG-mCherry (CmC), PGK-mCherry (PmC), EF1α-mCherry (EmC), and CMV-mCherry (CMmC) by inserting mCherry into multiple cloning sites. The constructs encoding the full-length amyloid precursor protein (APP) with the K670M/N671L (Swedish), I716V (Florida), and V717I (London) mutations (APPSweFlLon) and presenilin-1 (PSEN1) with the M146L and L286V mutations (PSEN1M146L/L286V) were cloned from the brain tissue of 8-month-old 5XFAD mice (B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799Vas/Mmjax; The Jackson Laboratory;http://www.jax.org/strain/006554). The cloned sequences of APP and PSEN1 harboring the fAD mutations from 5XFAD mice are of human origin. Total RNA was extracted using an RNA Extraction Kit (Qiagen) and reverse transcribed using a cDNA synthesis kit (Bio-Rad). APPSweFlLon and PSEN1M146L/L286V cDNA were PCR-amplified with restriction enzymes using KOD-Plus-Neo (Toyobo). The primers used for cloning were: APP-BamH I-F, 5′-GAATGGATCCATGCTGCCCGGTTTGGCACTG-3′ and APP-Mlu I-R, 5′-GAATACGCGTCTAGTTCTGCATCTGCTCAAA-3′; PSEN1-BamH I-F, GAATGGATCCATGACAGAGTTACCTGCACCG-3′ and PSEN1-Mlu I-R, GAATACGCGTCTAGATATAAAATTGATGGAA-3′. Next, APPSweFlLon and APPSweFlLon-IRES-PSEN1M146L/L286V, which were constructed using the amplified APPSweFlLon and PSEN1M146L/L286V genes, were inserted into multiple cloning sites to obtain two additional constructs: CAG-APPSweFlLon (CA) and CAG-APPSweFlLon-IRES- PSEN1M146L/L286V (CAP). All newly constructed vectors were confirmed by sequencing (Enzynomics).

CA or CAP lentiviral particles were produced by cotransfection of each plasmid with the packaging plasmids (an envelope plasmid harboring VSVg gene and another plasmid harboring the gag-pol genes) into 85% confluent HEK293T cells (ATCC) in a 10-mm tissue culture plate using the FuGENE® HD transfection reagent (Roche). The medium was changed at 24 h after transfection, and 10 mL of virus-containing medium was harvested once a day for three days. Each aliquot of the collected medium (30 mL) was concentrated to 200 µL by ultracentrifugation (25,000 rpm) at 4 °C for 2 h (Hitachi). Next, feeder-free cultured hPSCs (hESCs or hiPSCs) in a well of the 12-well tissue culture plate were transfected with CA or CAP viral particles by changing the medium to Essential-8 medium containing 2 × 107 IU/mL concentrated viral particles and 2 µg/mL hexadimethrine bromide (Sigma-Aldrich). The medium was changed every day for three days. Five hundred hPSCs transfected with CA or CAP viral particles were plated into the STO-plated well of a 6-well plate (Corning). After 7 days of culture, every cell-derived colony was manually dissected and transferred into the wells of a Matrigel-coated 12-well culture plate. After expansion under feeder-free conditions, the expression levels of APP and PSEN1 (CTF) were analyzed by western blotting.

The hESCs transfected with the CA or CAP lentiviral particles were named the CA hESC line and CAP hESC line, respectively, and the hiPSCs transfected with the CAP lentiviral particles were named the CAP hiPSC line. Each cell line was imaged using an inverted microscope in bright-field or fluorescence mode (DM IL LED Fluo; Leica).

Whole-genome sequencing

Whole-genome sequencing of the CAP hESC line was performed using the Illumina NovaSeq 6000 at a mean coverage depth of 30×. BWA-mem v.0.7.17 [14] was used with the default options to map the raw reads of 151 bp to the human reference genome (GRCh38) with the CAP lentiviral vector sequence. The resulting alignment file in the BAM format was sorted using Samtools v.1.16.1. Chimeric reads aligned to both vector and flanking human genome sequences allowed us to locate the vector integration site.

Single-cell RNA sequencing (scRNA-seq)

Raw reads from the scRNA-seq analysis were processed with Cell Ranger v.7.1.0 (10X Genomics) using the human reference genome (GRCh38) to generate the unique molecular identifier count matrices. Normalization and cell-type clustering were performed using the Seurat v.5.0.1 package in R (v.4.2.2) [15]. High-quality cells were selected based on the number of genes detected (200–2,500), and cells with high mitochondrial counts (> 10%) were excluded. The FindClusters function was used to identify cell clusters (resolution 0.65), and the cells were clustered and visualized using the UMAP method [16].

CO culture

COs were generated using a previously established protocol [17]. In brief, embryoid bodies (EBs) were formed by plating 9,000 feeder-free cultured hPSCs (hESCs or hiPSCs) dissociated into single cells into the wells of 96-well ultra-low-attachment plates (Corning) containing hPSC medium supplemented with 50 µM of Y27632, a ROCK inhibitor (Tocris), and 5 ng/mL of bFGF (day 0). The hPSC medium was replaced every other day for six days. On day 6, each EB was transferred to a 24-well ultra-low-attachment plate containing 500 µL of neural induction medium (1% MEM-NEAA, 1% GlutaMAX™, 1% N2 supplement (Thermo Fisher Scientific), and 1 µg/mL of heparin (Sigma-Aldrich) in DMEM/F-12). On day 8, 500 µL of fresh neural induction medium was added to each well. On day 10 of the protocol, the EBs were embedded in 20 µL droplets of Matrigel (BD Bioscience) and incubated in a gel for 1 h at 37 °C. These droplets were transferred and grown in CO differentiation medium without vitamin A (0.5% MEM-NEAA, 1% Glutamax, 1% B27 supplement without vitamin A, 0.5% N2 supplement, 2.5 µg/mL human insulin (Roche), and 3.5 µL/L β-mercaptoethanol in a 1:1 mixture of neurobasal medium (Thermo Fisher Scientific) and DMEM/F-12). The medium was changed on day 12, and on day 14, the Matrigel droplets were moved to a spinner flask containing CO differentiation medium (0.5% MEM-NEAA, 1% GlutaMAX™, B27 supplement (Thermo Fisher Scientific), 0.5% N2 supplement, 2.5 µg/mL human insulin, and 3.5 µL/L β-mercaptoethanol in a 1:1 mixture of neurobasal medium and DMEM/F-12). Thereafter, the medium was replaced every seven days.

Neuronal differentiation

Single cell-dissociated hESCs (1 × 105) were plated onto Matrigel-coated 24-well plates in mTeSR™1 supplemented with 5 µM of Y27632 (day − 4). On day − 3, the medium was replaced with mTeSR™1 without Y27632, and the medium was changed daily. On day 0, the mTeSR™1 medium was replaced with neuronal cell induction medium (1% MEM-NEAA, 1% GlutaMX™, 2% B27 supplement, 1% N2 supplement, and 20 µg/mL of human insulin in a 1:1 mixture of neurobasal medium and DMEM/F-12), and the medium was replaced every day until day 10. On day 11, the dissociated cells were resuspended in a neuronal cell induction medium supplemented with 5 µM of Y27632 and replated onto a Matrigel-coated 24-well plate (3 × 105 cells/well). On day 13, the medium was changed to neuronal cell induction medium supplemented with 100 nM of LDN-193,189, 10 µM of SB431542, and 2 µM of XAV 939. The medium was refreshed every other day until day 17. On day 19, the medium was changed to a neuronal cell maturation medium (1% N2 supplement, 2% B-27 supplement, and 25 ng/mL of BDNF in neurobasal medium). The medium was then changed every other day. On day 25, the cells were dissociated and replated onto a Matrigel-coated 24-well plate (1 × 105 cells/well), and the medium was replaced every other day.

Immunocytochemistry

The dissociated cells were fixed with a 4% paraformaldehyde (PFA) solution in PBS (Wako). Cells were permeabilized and blocked with 10% normal goat serum (NGS) (Vector) in 0.1% PBST (vol/vol, Triton X-100 in PBS). The cells were incubated with primary antibodies in PBST containing 2% NGS, and the following primary antibodies were used against the proteins: Nanog (rabbit, Cell Signaling), Oct4 (mouse, Santa Cruz), Tra-1-60 (mouse, Santa Cruz), SSEA4 (mouse, Santa Cruz), and PHF-tau (mouse, BioLegend). After three washes with PBST, the cells were incubated with the goat antirabbit or goat antimouse Alexa Fluor™ 488 conjugated (Thermo Fisher Scientific) secondary antibodies. Images were obtained using confocal microscopy (Leica TCS SP5 II; Leica).

Immunohistochemistry

The COs were fixed with a 4% PFA solution in PBS. The COs were allowed to sink in 15% and 30% sucrose solutions sequentially, followed by embedding in an optimal cutting temperature compound (Leica). The COs were then cryosctioned into 15-µm-thick slices using a cryotome (CM1850 cryostat; Leica). For immunohistochemistry, the sections were permeabilized and blocked with 10% NGS in PBST, and the sections were incubated with primary antibodies in PBST containing 2% NGS. Primary antibodies against the following proteins were used: 6E10 (mouse, BioLegend), 4G8 (mouse, BioLegend), PHF-tau (mouse, BioLegend), TUJ1 (rabbit, Cell Signaling), SOX2 (rabbit, Cell Signaling), TUJ1 (mouse, R&D Systems), TBR2 (mouse, R&D Systems), DCX (rabbit, Cell Signaling), PAX6 (rabbit, BioLegend), MAP2 (rabbit, Cell Signaling), MAP2 (mouse, Abcam), and N-cadherin (rabbit, Santa Cruz). After washing three times with PBST, the tissues were incubated with goat antirabbit or antimouse Alexa Fluor™ 488 and 647 conjugated (Thermo Fisher Scientific) secondary antibodies. Images were obtained using confocal microscopy (Leica TCS SP5 II; Leica).

Amylo-Glo staining

Amylo-Glo staining was performed using the Amylo-Glo RTD® Amyloid Plaque Stain Reagent (Biosensis). Briefly, the cryosection slides were washed three times with distilled water (DW) and transferred to 70% ethanol at room temperature for 5 min. The slides were then rinsed in DW for 2 min without shaking. The slides were then incubated for 10 min in the prepared 1× staining solution and rinsed with PBS for 5 min without shaking. The slides were then briefly rinsed in fresh DW and mounted with coverslips using a mounting medium. Images were obtained using confocal microscopy (Leica TCS SP5 II; Leica).

Thioflavin-S staining

The cryosection slides were washed three times with DW and incubated in 1% aqueous thioflavin-S (Sigma-Aldrich) solution (v/v in DW) for 15 min. The slides were then washed twice with 80% ethanol for 3 min before washing with 95% ethanol for 3 min. The slides were then briefly rinsed in fresh DW and mounted with coverslips using mounting medium. Images were obtained using confocal microscopy (Leica TCS SP5 II; Leica).

Bielschowsky’s silver staining

Bielschowsky’s silver staining was performed using the VitroView™ Bielschowsky’s Silver Stain kit (VitroVivo Biotech). Briefly, the cryosectioned slides were incubated in a prewarmed (40 °C) silver nitrate solution for 15 min before rinsing with DW. The slides were incubated in ammonium silver solution at 40 °C for 30 min. The slides were placed in a Developer Stock Solution for 1 min, after which the reaction was halted by immersing the slides in a 1% ammonium hydroxide solution for 1 min. After three washes with DW, the slides were placed in a 5% sodium thiosulfate solution for 5 min before washing three times with DW. After dehydration with 100% ethanol, the slides were incubated in xylene, and the slides were then mounted with coverslips using a mounting medium. Images were obtained using a microscope (AXIO; Zeiss).

Western blot analysis

The COs or neuronal cells were sonicated in RIPA buffer (iNtRON Biotechnology) on ice (Vibra-Cell™). The proteins that were obtained from each sample were separated using 10–12% SDS-PAGE resolving gels and transferred to polyvinylidene fluoride (PVDF) transfer membranes (Millipore). The membranes were washed with TBST (150 mM NaCl, 10 mM Tris-HCl [pH 7.6], and 0.1% Tween-20), blocked with 5% skimmed milk (Millipore) for 1 h, and incubated with primary antibodies. Primary antibodies against the following proteins were used: PHF-tau (mouse, BioLegend), Tau (mouse, Cell Signaling), amyloid precursor protein (APP) (mouse, Cell Signaling), presenilin (PSEN)-1 (rabbit, Cell Signaling), and β-actin (mouse, Santa Cruz). The membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-linked goat antirabbit (Santa Cruz) or goat antimouse (Santa Cruz) IgG secondary antibodies. An enhanced chemiluminescence solution (Thermo Fisher Scientific) was used to visualize the bands. Images were obtained using the Chemidoc™ imaging system (Bio-Rad). Densitometric analysis was performed using ImageJ software (National Institutes of Health (NIH)).

Enzyme-linked immunosorbent assay (ELISA)

The Aβ 1–40 and Aβ 1–42 levels were determined using commercial human amyloid-β assay kits (IBL). Samples were obtained from each CO sonicated in RIPA buffer on ice, and the sample proteins were quantified using the BCA method, and ELISA was performed using 10 µg of lysate, according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader (Infinite M200 Pro, Tecan).

Flow cytometry

Dissociated cells were fixed with a 4% PFA solution in PBS. The cells were blocked and permeabilized with 10% NGS in PBST for 1 h at room temperature. The cells were then incubated with the primary antibody in PBST containing 2% NGS. After washing three times with PBST, the cells were incubated with secondary antibodies for 3 h at room temperature. The samples were analyzed using flow cytometry (FACS Aria III, BD Biosciences).

Statistical analysis

All results are expressed as the mean ± standard error of the mean (SEM). Differences between the mean values were analyzed using the Student’s t-test and were considered statistically significant at P < 0.05.