Human-induced pluripotent stem cells and culture

KYOU-DXR0109B (ACS-1023; ATCC) and IPSC1030 (iPSC β-ACTIN GFP; Sigma-Aldrich) hu-iPSCs were routinely cultured on low growth factor Matrigel (Roche) in mTeSR Plus medium (Stem Cell Technologies) with 5% CO2 at 37 °C in a humidified incubator as described in the mTeSR handbook. Colonies were passaged at approximately 70–80% confluency before colonies had started to contact each other.

Human cerebral organoid generation and routine culture

COs were generated using Stem Cell Technologies cerebral organoid kit. Briefly, for embryoid body (EB) production half of a 70–80% confluent 25 cm2 flask of hu-iPSCs was used to seed a 96-well plate. Cells were plated in 50 µl EB media and additionally supplemented on day 0 with 50 μM Y27632. EBs were fed an additional 50 μl of EB media on days 2 and 4. EBs were transferred into neural induction medium (1× glutamax, 1× non-essential amino acids, 1% [v/v] N2, and 1 μg/ml heparin in DME-F12 medium) in low adhesion plates on day 5. Organoids were embedded in Matrigel (Corning) 2 days after neural induction when neuroectoderm was visible. Matrigel embedding was performed using organoid embedding sheets with 20 μl of Matrigel and allowed to set for 30 min at 37 °C. Organoids were then washed into a 6 well low attachment plate and incubated in cerebral organoid maintenance media (1× glutamax, 1× penicillin–streptomycin solution, 0.5× non-essential amino acids, 0.5% [v/v] N2, 1 μl/4 ml insulin, and 1 μl/286 ml 2-Merceptoethanol in 1:1 Neurobasal:DME-F12 medium) with 1% (v/v) B27 minus retinoic acid. On day 10, organoids were transferred into cerebral organoid maintenance medium with 1% (v/v) B27 plus retinoic acid for long-term culture. Long-term agitated culture was performed in 125-ml Erlenmeyer flasks on an orbital shaker at 85 rpm. Media was changed 1–2 times weekly.

NPC differentiation and NPC transplanted to COs

Neuronal Progenitor Cells were generated using Neural Induction Kit with SMAD inhibitors (Stem Cell Technologies). Briefly, iPSC cells expressing GFP were plated at 1.3 × 106 with neural induction media plus SMADi. Cells were grown and passaged 3 times in neural induction media with SMADi. After the third passage, cells were frozen in neural induction media with SMADi and 10% DMSO until experiments were started. 11 days prior to embedding COs NPCs were thawed and passaged one additional time in neural induction media with SMADi.

NPCs were collected using accutase and centrifuged at 300 × g for 5 min. Cells were then counted using the Muse cell count and viability reagent a Muse flow cytometer (Guava Instruments), which showed viability to be ~ 92%. Cells were mixed with Matrigel to provide 100,000 viable cells per 20 µl with a 1:2 ratio of cell media to Matrigel. Organoids were transferred to organoid embedding sheets and extra media was removed. Twenty µl of cell/ Matrigel mixture was then pipetted onto to each organoid. Organoids were rolled in Matrigel using a pipette tip to coat and incubated at 37 °C for 20–30 min to allow the Matrigel to solidify after which time organoids were then transferred back into flasks in organoid maintenance media with vitamin A. Organoids not receiving NPC treatment were embedded in a Matrigel/media mix void of cells using the same protocol described above.

Prion infections of human COs

Brain homogenates from sporadic CJD subtype MV2 or a normal brain homogenate (NBH) were diluted into organoid maintenance media to a final concentration of 0.1% (tissue wet weight/volume). At the start of infection, existing media was removed from the organoids and replaced with the inoculated media. Twenty-four hours after inoculation, an equivalent volume of fresh media was added to the cultures (diluting the original inoculum 1 in 2). A full media and culture vessel exchange was performed 7 days after initial exposure. Organoids were maintained in agitated culture with weekly media changes. All brain tissues used in this study were obtained on autopsy and were therefore exempt from review by the NIH Office of Human Subjects Research Protection.

Prestoblue analysis and LDH

Prestoblue metabolism was measured as per the manufacturer’s instructions from 4 to 6 random representative organoids from each group prior to infection, prior to NPC treatment, 2 weeks following treatment and at the conclusion of the study. Briefly, prestoblue reagent was diluted 1 in 10 in organoid media. Existing organoid media was removed and organoids were incubated in prestoblue-containing media for 30 min. The metabolized prestoblue-containing media was then transferred into replicate wells for analysis. Prestoblue fluorescence was measured at 560 nm excitation and 590 nm emission in a ClarioStar plate reader (BMG). Lactate dehydrogenase (LDH) was measured from 12 organoids per group at the ~ 180 dpi timepoint. Twenty-four hours prior to LDH measurement, organoids were separated into 24 well plates with 0.5 ml of fresh media per organoid/per well. One hundred µl of this 24-h old media was then mixed with 100 µl of LDH, dye and catalyst, incubated at 37 °C for 15 min and developed with 50 µl LDH stop solution, before absorbance was measured on the ClarioStar plate reader (BMG) at 460 nm (with reference wavelength 690 nm subtracted from the reading).

RT-QuIC

Real-time QuIC (RT-QuIC) assays were performed similarly to those reported previously [21]. Briefly, the RT-QuIC reaction mix contained 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/ml hamster recombinant PrP 90–231, 10 μM thioflavin T (ThT), 0.002% SDS (from the homogenate dilution), and 1 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA). Organoids were homogenized by motorized pestle to 10% (w/v) in PBS and cleared with a 2000 × g 2 min centrifugation. Organoid homogenates were serially diluted in 0.1% SDS/PBS/N2 solution, and 1 μl was loaded into each well of a black 384-well plate with a clear bottom (Nunc) containing 49 μl of reaction mixture. Plates were sealed (Nalgene Nunc International sealer) and incubated in a BMG FLUOstar Omega plate reader at 50 °C for 50 h with cycles of 60 s of shaking (700 rpm, double-orbital) and 60 s of rest throughout the incubation. ThT fluorescence measurements (excitation, 450 ± 10 nm; emission, 480 ± 10 nm [bottom read]) were taken every 45 min. Spearman–Kärber analyses was used to provide estimates of the concentrations of seeding activity units giving positive reactions in 50% of replicate reactions, i.e., the 50% “seeding doses” or SD50’s as previously described [21].

Proteinase-K digests and western blotting

10% organoid homogenates were treated with 10 μg/ml Proteinase-K in 1% Sarkosyl for 1 h at 37 °C with 400 rpm shaking. The reactions were stopped by incubation with 1 μM Pefabloc for 5 min at 4 °C. Samples were then mixed with 2 × Bolt LDS sample buffer (Invitrogen) containing 6% β-mercaptoethanol and boiled for 5 min. For western blots that were not PK treated, samples were mixed with sample buffer and boiled for 5 min. Samples were run on Bolt 4–12% Bis–Tris gels (Invitrogen) and transferred to PVDF membranes using the iBlot 2 transfer system (Invitrogen). Antibodies were used at the following concentrations, PSD-95 1:5000, doublecortin (Abcam) 1:2000, sox2 (Cell Signaling Technologies) 1:5000, oxidative stress cocktail 1:5000, GFAP (Abcam) 1:2000, 3F4 1:5000 (Millipore), AKT (Cell Signaling Technologies) 1:1000. Secondary antibodies form Abcam were goat anti-mouse 1:5000 and goat anti-rabbit 1:5000. Bands were visualized using SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate (Invitrogen) and imaged on the iBright imaging system (Invitrogen). Blots were normalized as a ratio of target to total protein using Coomassie stain, and quantification was preformed using Image J.

Multi-electrode arrays (MEA) recording

COs were adhered to the MEA using 0.005–0.01% polyethyleneimine (PEI) and 2 μg/mL laminin as described previously (PMID: 32,976,764). Each well of 24-multi-well MEA (multi-channel systems) was pre-coated with 100 µL PEI for 1 h at room temperature, washed three times with MilliQ water (5 min/wash), and allowed to air dry. Once dried, the wells were coated with Laminin by adding 20 µL of 2 µg/mL Laminin (Corning) to each well to cover all electrodes and incubating in Laminin for ~ 1 h at room temperature. The wells were washed three times with MilliQ water and filled with ~ 800 µL BrainPhys media (Stem Cell Technologies). The COs were then dropped into pre-coated wells (about one organoid/well) and gently moved to the center of the well to ensure good contact with the electrodes. Plated COs were incubated without shaking in a 5% CO2 incubator for at least 18 h before reading the neuronal network activity by a multiwell MEA system (multi-channel systems). Local field potential was recorded at a sampling frequency of 20 kHz and filtered using a second-order Butterworth high-pass filter (300 Hz) and a fourth-order Butterworth low-pass filter (3500 Hz). Spikes were detected as local field potential peaks (positive or negative) greater than 3.6 standard deviations of the mean local field potential noise level. A cluster of spikes from a single electrode was considered a burst when at least four spikes were detected within 100 ms. A network burst was detected when at least three electrodes showed overlapping bursts. An electrode detecting less than 250 spikes per minute was considered inactive.

Immunohistochemistry and immunofluorescence

Five COs from each experimental group were submitted for histologic studies. Organoids were immersed in 3.7% neutral buffered formalin for ~ 24 h prior to standard embedding in paraffin. Five-micron sections were cut, stained and examined by routine H&E staining for overall pathology. IHC staining specifically for prion protein was performed using anti-PrP antibody SAF32 (Cayman Chemical) [26]. De-paraffinization, antigen retrieval and staining were performed using the Discovery Ultra-Staining Module. Antigen retrieval for SAF32 staining was achieved using extended cell conditioning with CC1 buffer (Ventana) containing Tris–Borate-EDTA, pH 8.0 for 64 min at 95 °C. Prior to staining, a horse serum blocker (Vector #136,021) was applied at 37°C for 20 min. To stain PrP, we applied SAF32 at a dilution of 1:2,000 in antibody dilution buffer (Ventana) for 1 h at 37 °C. The secondary antibody, horse anti-mouse IgG (Vector#30,129) was applied undiluted for 32 min at 37 °C. Detection was performed with ChromoMap DAB (Roche/Ventana #NC1859896). All histopathology slides were analyzed and photographed Aperio Imagescope software.

NPCs for characterization were grown overnight in a chamber slide before being fixed with 3.7% neutral buffered formalin for 15 min. Chamber slides were then rinsed with PBS before staining with Sox2 (Cell Signaling Technologies) 1:1000, Doublecortin (Abcam) 1:2000, NFL (Santa Cruz Biotechnology) 1:100, and Dapi 1:10,000. NPC characterization cells were then counted and expressed as a ratio of positive target cells to Dapi from the same field.

Organoid Map2 staining was performed on de-paraffined, antigen retrieval sections using the same protocol as above. Map2 (Synaptic Systems) was used at 1:200. GFP visualization of NPCs were done on live organoids. All fluorescent imaging was performed with an EVOS microscope using the same exposure settings between groups and quantification was done using ImageJ. Map2 quantification was from one 4× image per organoid with five organoids represented per group and was normalized to size of organoid.

General CO characterization was performed on 6- to 11-month-old organoids. Immunohistochemistry fixation, embedding and sectioning were performed as described above. IHC staining for neuronal and astrocyte markers were preformed using the following dilutions: Sox2 (Cell Signaling Technologies) 1:200, doublecortin (Abcam) 1:5000, NFL (Santa Cruz Biotechnology) 1:500, Map2 (Abcam) 1:2500, GFAP (Dako) 1:3500. Appropriate secondaries for IHC where undiluted either horse anti-mouse or horse anti-rabbit (Vector). Immunofluorescence were performed on frozen cryosections and imaged using a confocal. Primary antibodies, NFL, B Tubulin, and F-actin were used at 1:200 and DAPI was 1:10,000. Appropriate secondary antibodies were used at 1:500.

Bioplex

Organoids were triturated in 200 µl cell lysis buffer including PMSF and factor QG (BioRad). Samples were then frozen until later analysis were performed. Once thawed samples were cleared of debris by centrifuging at 15,000 × g for 10 min at 4 °C. Cleared samples were then analyzed using a BCA assay and further diluted in cell lysis buffer to ensure all samples were in a range appropriate for the BioPlex cell signaling assays, around 200 µg/ml. BioPlex cell signaling panels MAPK 9-plex and AKT 8-plex panels were run according to BioPlex instructions on the BioPlex 3D suspension array system. Mean fluorescents for each analyte were normalized to total protein as determined by an average from 2–3 independent BCA runs.