Control lines used for cerebral organoid generation were described previously . Isogenic iPSC with MAPT mutations were generated by Janssen and obtained from EBiSC (https://ebisc.org)(19). iPSC with the following MAPT genotypes were used and the EBiSC accession numbers are given in parentheses:WT (SIGi001-A-1), 10+16m mono-allelic (SIGi001-A-13), 10+16bibiallelic (SIGi001-A-12), and 10+16 biallelic/P301S biallelic (SIGi001-A-10).
iPSC lines were cultured in feeder-free conditions on plates coated with Geltrex (Gibco, A14133-02) diluted 1:100 with DMEM/F12 (Gibco, #10565-018). Cultures were fed daily with Essential 8 media (Gibco, #A15169-01, + Supplement, Gibco, #A15171-01) and passaged every 5–7 days using 500µM EDTA (Invitrogen, #15575-038).
Differentiation of iPSC into 2D cortical neurons
Differentiation of iPSC into cortical neurons was performed as described in Shi et al (Shi et al, 2012). Briefly, iPSC lines were grown to 100% confluence before switching to neural induction media, which is N2B27 neuronal maintenance media containing 10μM SB431542 (Tocris) and 1 μMDorsomorphin (R and D systems,#1614/10). N2B27 neuronal maintenance media consisted of a 1:1 mixture of Dulbecco's modified eagle medium F12 and Neurobasal (Gibco, #12348-017) supplemented with 0.5% N2 (Gibco, #17502-048), 1% B27 (Gibco, #17504044), 0.5% non-essential amino acids (NEAA) (Sigma, #M7145), 1 mM L-glutamine (Gibco, #25030081), 25U pen/ strep (Gibco, #H4034-100G), 10 μM β-mercaptoethanol (Gibco, #31350-010) and 25U insulin (Sigma, #19278-5ML). Neural induction media was replaced daily, and 10-12 days post-induction the neuroepithelial layer was detached using dispase (Invitrogen,#17105-041), re-plated onto laminin-coated wells (Sigma, #L2020, 1:100 diluted in PBS, Gibco, 10010023) in N2B27 maintenance media without SB43142 and Dorsomorphin. Media was replaced every 3 days and precursors were passaged using dispase onto fresh laminin-coated plates every 5-7 days. The final passage was performed at day 35 using accutase (Gibco #A11105-01), and cells were plated at a final density of 50,000 cells per cm2 onto plates coated with poly-ornithine (Sigma, #P4957) and laminin, and maintained in N2B27 media until the required timepoint.
Differentiation of iPSC into 3D cerebral organoids
Cerebral organoids were generated using the protocol described by Lancaster et al (27). Briefly, iPSC were detached from plates using EDTA, prior to dissociation into single cells using accutase. Single cells were plated into low attachment round bottomed 96 well plates (CoStar, #7007) at a density of 9000 cells per well in low FGF/standard organoid hESC media: DMEM F12 with the addition of 10% knock out serum replacement (Thermo #10828028), 3% embryonic stem cell quality fetal bovine serum (ESC-Quality FBS) (Invitrogen, #10828-028), 1% GlutaMAX (Invitrogen, #35050-038) , 1% non-essential amino acids (NEAA) and 0.1 mM β-mercaptoethanol, bFGF (Peprotech, #100-18B) at 4ng/mL and 50mM Y-27632 ROCK inhibitor (VWR, #688000-5). Embryoid bodies were fed every 48h. At day 6 the embryoid bodies were transferred to low attachment 24 well plates (Corning, #3473) and induction media added: DMEM F12 with the addition of 1% N2 supplement, 1% NEAA and Heparin (Sigma, #H3149-25KU) to a final concentration of 1µg/mL.
At day 10-12 the neuroepithelial tissues were embedded in 20-30µL of Matrigel (BD Biosciences, #356234) using parafilm moulds and allowed to polymerise at 37°C. The embedded organoids were then placed in a 5cm tissue culture dish (Corning, #430589) containing Cerebral Organoid Differentiation Media (CODM) without Vitamin A: 1:1 ratio of DMEM-F12 Medium and Neurobasal Medium, 0.5% N2 supplement, 0.025% Insulin, 1% GlutaMAX supplement, 0.5% NEAA, 0.5% Penicillin Streptomycin (Gibco, #H4034-100G), 50mM β-mercaptoethanol, 2% of B27 supplement without Vitamin A (Gibco, #12587010).
The media was replaced after 48h with CODM with the addition of vitamin A within the B27 supplement and moved onto an orbital shaker at 55-85 rpm (IKA, #0009019200). Once transferred to the shaker, media was replaced every 3-4 days until the required time point. Phase contrast images of organoids were taken using an Olympus CKX41 phase contrast microscope.
Engineered Cerebral Organoids (enCORs) using iPSC and PLGA fibers
Engineered cerebral organoids (enCORs) were generated according to the protocol described by Lancaster et al (24) . PLGA Vicryl violet sutures (Ethicon, #W9567) were cut into pieces <1mm in length. 5-10 fibres were placed in each well of a 96 well round bottomed low attachment plate. 18,000 iPSC cells (prepared as described above) were seeded per well on top of the fibres. Embryoid bodies were maintained as described above, embedded in Matrigel at day 11-12 but kept in induction media from days 5/6 to days 12/13. Two days post-embedding, the media was changed to Improved Differentiation Media minus vitamin A (IDM-A): 1:1 ratio of DMEM-F12 Medium and Neurobasal Medium, 0.5% N2 supplement, 0.025% Insulin, 1% GlutaMAX supplement, 0.5% NEAA, 0.5% Penicillin Streptomycin, 50mM 2-mercaptoethanol, 2% of B27 supplement without vitamin A. On days 13-16 3mM CHIR99021 (Tocris, #4423) was added to the media.
At day 18-20 enCORs were moved to the orbital shaker at 85rpm, to give them a throw of 10mm. From day 20 onwards Improved Differentiation Media plus Vitamin A & C + HEPES (IDM+vA) was used: a 1:1 mix of DMEM-F12 Medium andNeurobasal Medium, 0.5% N2 supplement, 0.025% Insulin, 1% GlutaMAX supplement, 0.5% NEAA, 0.5% Penicillin Streptomycin, 50mM b-mercaptoethanol, 2% of B27 supplement with Vitamin A, 0.5µg/mL Vitamin C / Ascorbic acid (Sigma, #A4403-100MG), 1.49g HEPES per 500mL (Sigma, #H4034-100G). The media was changed every 3-4 days and 1% Matrigel was added to the media from day 40 onwards. Phase contrast images of organoids were taken using an Olympus CKX41 phase contrast microscope.
Generation of Forebrain Organoids
Forebrain organoids were generated according to the protocol described by Qian et al (28). Briefly, EBs were generated as described in the cerebral organoid protocol, with the addition of 2 μM SB431542 and 2 μMdorsomorphin to the embryoid body media as well as the omission of bFGF. Embryoid bodies were fed every 48h and were not transferred to 24-well plates remaining in the 96-well plates. On day 6 dual SMAD cerebral organoid induction media was added. Similar to the cerebral organoid induction media but with the addition of 1mM SB431542 and 1mM CHIR99021. At day 7 the neuroepithelial tissues were embedded in 20-30µL of Matrigel and embedded organoids were then placed in a 5cm tissue culture dish containing dual SMAD induction media.
The media was replaced at day 14 with CODM with the addition of vitamin A and moved onto an orbital shaker at 55-85 rpm. Once on the shaker the media was replaced every 3-4 days.
Fixation of organoids and preparation of frozen sections
Whole COs and enCORs were fixed in 4% paraformaldehyde for 30 min at RT, washed with PBS and immersed in 30% sucrose overnight at 4°C. Samples were embedded in optimum cutting temperature compound (OCT) and 10 µm sections cut using a cryostat.
Tissue sections were permeabilised using PBS 0.3% Triton X-100 (PBS-T), blocked with 5% bovine serum albumin (BSA) in PBS-T for 1 hour before incubation with primary antibodies (Table 2) diluted in blocking solution overnight at 4°C. Slides were then washed with PBS-T, incubated with secondary antibodies (Table 4) at 1:1000 concentration for 1h at RT. Following PBS-T washes, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma) for 5 mins and then washed with PBS-T. A coverslip was attached to the slide using Fluorescent Mounting Medium (DAKO, #53023) and stored at 4°C.
Slides were imaged using a Leica CTR6000 and Hamamatsu C10600 camera.
SDS-PAGE and Western Blotting
3D or 2D neuronal cultures were lysed in RIPA buffer (10mM Tris-CL, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140mM NaCl) or TRIS lysis buffer ( 25mM Tris-HCL, 0.8M sodium chloride, 1mM EGTA, 50mM imidazole, 25mM beta-glycerophosphate, 20mM sodium fluoride, 10mM sodium pyrophosphate, 0.5mM phenylmethylsulfonyl fluoride). Protease inhibitors (Roche, #11836170001) and phosphatase inhibitors (Roche, #04906845001) were added just before use.Protein concentrations were determined using BCA assay (Biorad), and 4x LDS loading buffer (Invitrogen, #NP0007) supplemented with 20% DTT was added to equal amounts of protein before heating at 98°C for 10 min and centrifugation for 3 minutes at 12000gav. Samples were electrophoresed on 4-12%- SDS-PAGE gels (Invitrogen, #NP0321BOX) at 150V for 3h in running buffer (MES NuPAGE SDS, NOVEX, #NP0002) prior to transfer onto nitrocellulose membrane (GE Healthcare, #10600003) in transfer buffer: H20 supplemented with 10% Tris/Glycine (Geneflow, #EC-880) and 20% methanol (VWR, #UN1230), for 1hr 15 mins at 30V, blocked in 5%-non-fat milk in PBS with agitation for 1h prior to overnight incubation at 4°C in primary antibody (Table 3) diluted in 5% milk in PBS-T. Membranes were washed with PBS-T before incubation for 1 hour with secondary antibodies (Anti-mouse Alexa Fluor, #A21058 & anti-rabbit Rockland, #611-145-122) diluted (1:20,000) in 5% milk in PBS-T. Secondary antibodies were visualised at 700nm and 800nm using the Li-Cor Odyssey Fc system
Dephosphorylation of protein lysates was performed as described previously(29). 10% 10X PMP buffer (New England Biolabs, #B07615), 10% 10mM MnCL2 (New England Biolabs, #B17615) and 4000U l-protein phosphatase (New England Biolabs, #P0753L) was added to equal amount of protein. Samples were vortexed gently for 10 seconds prior to incubation at 30°C for 3h. l-protein phosphatase was inactivated by the addition of LDS-buffer and processing for electrophoresis as described above.
Cells and organoids were lysed in 1mL of Trizol (Thermo15596026) and incubated at 4°C with agitation for 20 mins. RNA was extracted from these lysates according to the manufacturers protocol. Briefly, 0.2mL of chloroform was added to the sample, incubated at RT for 2-3 mins and centrifuged at 12,000g(av) at 4°C. Aqueous phase was collected and 0.5mL of isopropanol was added, incubated for 10 mins and centrifuged at 12,000g(av) for 10 mins at4°C. The supernatant was discarded and 1mL 75% ethanol was added and vortexed before centrifugation at 7,500g(av) at 4°C. The supernatant was removed and the pellet was air dried for 10 min at RT. RNA pellets were diluted in 20µL of RNase free water (Ambion, #AM9937) and stored at -80°C. RNA concentration and purity was determined by nanodrop.
1000ng of RNA was reverse transcribed using Super Script IV kit (SSIV, Invitrogen 18090010) according to manufacturer’s instructions. Briefly, 1000ng of template RNA was added to RNase free water,2.5mM random hexamers (Invitrogen, #100026484) and 10mM dNTP mix (Invitrogen, #55082, #55083, #55084, #55085) and heated at 65°C for 5mins and then cooled on ice for 1 min. After which SSIV buffer, 100mM DTT, RNaseOUT (Invitrogen, #100000840) and SSIV reverse transcriptase was added to the reaction, heated to 23℃ for 10 mins, incubated at 50-55℃ for 10 mins and finally inactivated by heating at 80°C for 10 mins. The cDNA was then stored at -20°C.
RNA (5mg) was converted to cDNA by PCR using the High-Capacity cDNA Reverse Transcriptase kit (Life Technologies). Taqman real-time PCR assays were utilized to quantify expression for the following genes: exon 10-MAPT (Hs00902312_m1); Total MAPT (Hs00902194_m1).Samples were run in triplicate with replicate samples analyzed in each plate to control for plate-to-plate variability. To avoid amplification interference, expression assays were run in separate wells from the neuronal housekeeping gene MAP2(Hs00258900_m1). Real-time data were analysed by the comparative CT method. Average CT values for each sample were normalized to the average CT values for the neuronal housekeeping gene MAP2. The resulting value was then corrected for assay efficiency. Samples with a standard error of 20% or less were subsequently analysed.
Polymerase chain reaction
50ng of cDNA used per reaction with GoTaq Green master mix (Promega #M7123). PCR Reactions were set up according to the manufacturers’ protocol. Briefly, 10µL of GoTaq, 9.6µL of H2O, 0.2µM of forward, 0.2µM of reverse primer (Table 1 for primers used and sequences), 50ng template cDNA was added to each reaction. PCR products were separated on a gel composed of 1-2% agarose (Sigma, #A9539) dissolved in 1X TBE (Sigma, #93290) with 1X GelRed (Biotium,#BT41003) alongside a 100 base pair ladder (Bioline, #H4-617110A) at 100V for 1h. PCR products were visualised at 600nm using the Li-Cor Odyssey Fc system.
Tau HEK biosensor cell assay
Tau RD P301S FRET Biosensor cells (ATCC® CRL-3275™) were cultured in DMEM + GlutaMAX (Gibco, #31966-021) with 10% FBS (Gibco, #11573397) and split every 3-5 days using trypsin (Gibco, #25200056). For seeding assays, 115,000 cells were plated on 13mm coverslips (VWR, #631-0148P) in 24 well plates (Thermo, #142485) and left to attach for 24h. Lipofectamine 3000 (Invitrogen, #L3000-001) was used in Opti-MEM (Gibco, 31985062) according to the manufacturers protocol to transfect the Tau RD P301S FRET Biosensor cells with 10ug of enCOR lysate, AD brain lysate, or liposome-only negative control. Cells were fixed with 4% PFA and mounted on to slides before being imaged on confocal microscope. Slides were imaged at 405nm for detection of FRET signal or YFP only at 525-550nm.
Densitometric data was obtained from immunoblots using Image Studio software from Licor. GraphPad Prism 8 software was used to implementall statistical analysis of the immunoblot densitometricdata. Information on the number (n) of values used as well as the statistical tests applied can be found in the figure legends.