CTR_M3_36S human induced pluripotent stem cell (iPSC) line was reprogrammed from keratinocytes obtained from a neurotypical male. Keratinocytes were reprogrammed by introducing a set of Sendai virus encoding human OCT4, SOX2, KLF4, and C-MYC (Yamanaka factors) using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher) according to the manufacturer’s instructions. The virus was a gift from Dr. Mahito Nakanishi (AIST, Japan).
Stem cell maintenance
Cells were regularly tested for mycoplasma and certified mycoplasma-free. iPSCs were maintained in Essential 8™ medium (Thermo Fisher) without antibiotics at 37°C, 5% CO2, 5% O2 in 6-well NUNC™ plates (Thermo Fisher) coated with Geltrex™ (Thermo Fisher). Passaging of iPSCs lines were done with Versene (EDTA) solution (Lonza) according to the manufacturer’s instructions. Passaging ratio for iPSC maintenance was kept between 1:6 and 1:18.
iPSC colonies approaching 80% confluence were passaged at 3:2 ratio on 6-well NUNC™ plates coated with Geltrex™ on D–2/–1 and maintained at 37°C, 5% CO2, 5% O2 for 24–48 hrs until they approached 100% confluence. Directed differentiation began on D0 by changing Essential 8™ medium to neural induction medium and incubating the cells at 37°C, 5% CO2, 20% O2. Neural induction lasted for 7 days. To prepare neural induction medium, N2:B27 was first prepared by mixing the N2 medium (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F–12 Ham (DMEM/F12) (Sigma Aldrich) supplemented with 1X GlutaMAX™ (Thermo Fisher) and 1X N–2 supplement (Thermo Fisher)) and the B27 medium (Neurobasal® medium (Thermo Fisher) supplemented with 1X GlutaMAX™ and 1X B–27 supplement (Thermo Fisher) or 1X B–27 without vitamin A supplement (Thermo Fisher)) at 1:1 ratio. The following small molecule inhibitors were added to N2:B27 to make the neural induction medium: 100 nM LDN193189 (Sigma Aldrich) and 10 µM SB431542 (Sigma Aldrich) for dual SMAD inhibition (DSi); 100 nM LDN193189, 10 µM SB431542, and 2 µM XAV939 (Sigma Aldrich) for dual SMAD inhibition plus Wnt/β-catenin inhibition (DS-Wi); and 100 nM LDN193189, 10 µM SB431542, 2 µM XAV939, and 1 µM Cyclopamine (LC Laboratories) for dual SMAD inhibition plus Wnt/β-catenin plus sonic hedgehog inhibition (DS-WHi). Neural induction medium was used from D0 to D7, and N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire directed differentiation period.
Neural passaging 1, 2, and 3 were performed with Accutase (Thermo Fisher) on D7, D12, and D15/16, respectively. Briefly, cells were washed with room temperature HBSS and treated with Accutase at 37°C, 5% CO2, 5% O2 for 3–4 minutes. Cold Accutase was used for neural passagings 1 and 2, and room temperature Accutase was used for neural passaging 3. Cells in Accutase were then collected with a P1000 pipette. Extra care was taken during neural passagings 1 and 2 where P1000 pipetting was done no more than 5 times when cells in Accutase were collected. Collected cells were then mixed with room temperature DMEM/F12 (twice the volume of Accutase used) so that Accutase could be deactivated, and centrifugation was performed twice to wash off the Accutase from cells. Centrifugation was done at 900 revolutions per minute (RPM) for 2 min during neural passaging 1 and 2, and at 1250 RPM for 2 min during neural passaging 3. After centrifugation, cells were plated on new 6-well NUNC™ plates coated with Geltrex™. Passaging ratios were 1:1 for neural passaging 1 and 2, and 2:3 for neural passaging 3. To ensure cell survival 10 µM Y–27632 (Sigma Aldrich), a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was mixed with the plating medium at each neural passaging and then removed after 24 hrs.
Genomic DNA was extracted from iPSCs using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions. The APOE locus containing the rs429358 and rs7412 SNPs was amplified with Taq DNA Polymerase (QIAGEN) according to the manufacturer’s instructions. The primers used for APOE genotyping were previously designed by Henderson and colleagues (11), and they are able to generate PCR products that can be visualised easily by gel electrophoresis after HhaI enzyme (Thermo Fisher) digestion. Amplified PCR products were digested with 1 unit of HhaI digestion enzyme and gel electrophoresis was performed using a 3% agarose gel containing 0.5 µg/mL ethidium bromide.
Gene expression analysis
Total RNA was extracted from D7, D12, D15/16, and D18/19 cells that were not used for neural passaging with TRIzol® reagent (Thermo Fisher) according to manufacturer’s instructions and eluted in 25–30 µL of diethyl pyrocarbonate (DEPC)-treated water. Reverse transcription of total RNA into complementary DNA was performed using SuperScript® III First-Strand Synthesis System (Thermo Fisher) according to the manufacturer’s instructions. For gene expression analysis, real-time quantitative polymerase chain reaction (qPCR) was performed using the HOT FIREPol® EvaGreen® qPCR Mix (Solis Biodyne) according to the manufacturer’s instructions. CT values of APOE were normalised to that of GAPDH, and relative expression of APOE across samples were quantified using the 2-ΔΔCt method where D7 sample was used as a reference for each differentiation lineage.
GraphPad Prism v8 was used for statistical analysis. The statistical significance of the mean differences between groups were analysed by one-way analysis of variance (ANOVA) followed by Bonferroni correction for multiple testing. The mean, standard error of measurement (SEM), and number of biological replicates are reported. P-value < 0.05 was considered significant to reject the null hypothesis that the differences observed between groups is due to random variation.