Materials

Cells

H9 hESCs feeder-dependent and feeder-independent (from Wicell Research Institute, cat. No. WA09), H9 hESCs derived Human NSCs (hNSCs) were purchased from Gibco (cat. No. N7800200).

Growth media and supplements

DMEM-F12 medium (cat. no. 11320082), StemPro® NSC SFM—Serum-Free Human Neural Stem Cell Culture Medium (cat. No. A1050901), knockout serum replacement (KOSR, cat. no. 10828–028), MEM nonessential amino acids (MEM-NEAA, cat. No. 11140050). N 2 supplement (cat no.17502048), B-27 supplement without vitamin A (cat. No. 12587010), B-27 supplement with vitamin A (cat. No. 17504044), glutamine/glutaMAX (200 mM, cat. No. 35050061, A2916801), Dispase (cat no. 17105–0412q), and Collagenase IV (cat. No. 17104019) were obtained from Invitrogen (USA). Fetal bovine serum (FBS, cat. No. 10438–018) and bovine serum albumin (BSA, BP9703–100) obtained from Fischer Scientific (Gibco, Invitrogen, USA). Heparin (cat. No. H3149), basic fibroblast growth factor (bFGF, F0291), and epidermal growth factor (EGF, E9644) were procured from Sigma-Aldrich (USA) while mTeSR1 medium (Cat. No. 05850) was procured from Stem cell Technologies (USA).

Chemicals and antibodies

METH (cat. No. M8750), 5′-bromo-2′-deoxyuridine (BrdU) (cat. No. B5002), beta-mercaptoethanol (cat. No. M7522), and primary antibody GFAP (anti-mouse, Sigma, G3893) were purchased from Sigma Aldrich (USA). Protein IP lysis buffer (cat. No. 87787) and protease and phosphatase inhibitors cocktail (Cat. No. A32955) was obtained from Pierce (USA) while Bio-Rad DC protein assay kit (Cat. No. 5000112) and chemiluminescence substrate (cat. No. 34580) were purchased from Thermo Scientific (USA). Optimal cutting temperature (OCT) compound (cat. No. 4583) was procured from Tissue-Tech (USA). Primary antibodies such as Ctip2 (anti-rat, cat. No. ab18465), Sox2 (anti-rabbit, ab97959), β-Tubulin III (anti-mouse, ab7751–100), and Nestin (anti-chicken, ab134017) were obtained from Abcam, (USA). Ki67 (anti-rabbit, Millipore AB9260), and PVDF membrane were obtained from Chemicon (Millipore, Billerica, MA, USA). Rabbit anti-β-actin (cat. No. 4970) and Nlrp1 (anti-rabbit, cat. No. 4990) primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), while mouse anti-BrdU (cat. No. sc-32323) primary antibodies was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488, 594, and 647 conjugated secondary antibodies were purchased from Molecular Probes (Invitrogen, USA). Anti-fade mounting medium with DAPI was obtained from Vector Labs (Vectashield, Vector Laboratories, CA).

Cell culture and organoid generation

All studies were conducted in accordance with approved IRB protocols by the University of California, San Diego. H9 hESCs (WA09) from WiCell was cultured on a feeder layer of irradiated mouse embryonic fibroblasts or under feeder-free matrigel coated conditions following previously established protocols. Feeder-dependent H9 hESCs were detached from their feeder layer using 1 mg/ml collagenase (dissolved in DMEM-F12 medium) for 15–20 min at 37 °C in CO 2 incubator and 0.5 mg/ml dispase (dissolved in DMEM-F12 medium) for an additional 15 min at 37 °C in CO 2 incubator. Wells were washed with media to collect floating undifferentiated hESCs and colonies were dissociated using Accumax or Versene solution at 37 °C for 10 min to generate a single-cell suspension. At day 0, embryoid bodies were formed using the hanging drop method with 4500 cells/drop in DMEM/F12 media supplemented with 20% knockout serum replacement (KOSR), bFGF (4 ng/ml), MEM nonessential amino acids (MEM-NEAA, 1%, vol/vol), and glutamine (200 mM, 1%, vol/vol) or grown in microwell plates. After 2 days of hanging drop culture, embryoid bodies were transferred to sterile petri dishes with refreshed media. After 6 days in culture, embryoid bodies were transferred to new petri dishes containing neural induction media consisting of DMEM/F12, N2 supplement (1%, vol/vol), MEM-NEAA (1%, vol/vol), glutamine (200 mM, 1%, vol/vol), and heparin (1 μg/ml) until day 11. At day 11, organoids were transferred to Matrigel droplets (30 μl) and cultured in 1:1 mixture of DMEM/F12 and Neurobasal medium supplemented with B-27 without vitamin A (1%, vol/vol), N2 (1%, vol/vol), NEAA (1%, vol/vol), insulin, beta-mercaptoethanol, and glutamine (200 mM, 1%, vol/vol). Twenty organoids were then transferred to stir flask bioreactors (125 ml) containing magnetic shaft and stirring speed was maintained 50–60 rpm. For long term growth on day 15 in 75–100 ml of cerebral organoid differentiation media with vitamin A. Media was changed every 3 days. Organoids were treated with 5 μM METH dissolved in organoid culture media every other day for 1 week before fixing for immunostaining or dissociation for scRNA-seq.

NSC culture

Human NSCs derived from H9 hESCs were purchased from Gibco (N7800200) and cultured in Knockout D-MEM/F-12 media containing 2 mM GlutaMax (1%, vol/vol), 20 ng/ml bFGF, 20 ng/ml EGF, and 2% StemPro Neural Supplement (vol/vol) on Matrigel- or CELLStart-coated plates.

Astrocyte differentiation

NSCs were plated on Matrigel coated plates in complete Stem-Pro NSC SFM medium at 2.5 × 104 cells/cm2. After 2 days of NSC culture, the media will be changed to astrocyte differentiation medium which consists of D-MEM supplemented with N-2 (1%, vol/vol), GlutaMAX-I (2 mM, 1%, vol/vol), FBS (1%, vol/vol), and Antibiotic-Antimycotic solution (1%, vol/vol) every 3–4 days for 1 month.

Neuronal differentiation

To differentiate NSCs into the neurons, hNSCs were plated in polyornithine and laminin-coated culture dishes in complete StemPro NSC SFM at 2.5–5 × 104 cells/cm2. After 2 days, the media was changed to neural differentiation medium consisting of neurobasal medium supplemented with B-27 serum-free supplement (1×, vol/vol), GlutaMAX-I (200 mM, 1×, vol/vol), and antibiotic -antimycotic (1×, vol/vol) solutions and every 3–4 days for 2 weeks.

Immunohistochemistry

To section and stain organoids, organoids were washed with phosphate buffer saline (PBS) and incubated in cell recovery solution for 30 min to dissolve the surrounding Matrigel. Cell recovery solution is used to recover cells or organoids from matrigel matrix for subsequent sectioning and biochemical analyses. Organoids were washed with PBS and then fixed with 4% paraformaldehyde (PFA) for 1 h. Fixed organoids were washed three times with PBS, stained with hematoxylin for 5 min and incubated in 30% sucrose overnight. Sucrose solution was removed, organoids were washed with PBS and embedded in OCT compound for cryosectioning. We typically cut 20-μm-thick sections of organoid for immunohistochemical analysis using cryostat. Cryosections were blocked in 5% BSA in PBS for 1 h, washed three times with PBS + 0.1% Tween-20 (PBST), and incubated at 4 °C overnight with primary antibodies: Ctip2 (1:500, Rat, Abcam ab18465), Sox2 (1 μg/ml, Rabbit, Abcam ab97959), GFAP (1:500, Mouse, Sigma, G3893), β-Tubulin III (1:500, mouse, Abcam ab7751–100), Ki67 (1:200, Rabbit, Millipore AB9260), Nestin (1:1000, Chicken, Abcam ab134017), and Nlrp1(1:200, Rabbit, Cell Signaling Technology (CST)-4990). Cryosections were washed three times with PBS with 0.1% Tween-20 (PBST) to remove primary antibody 1 h prior to secondary antibody incubation. We used alexa fluor conjugated secondary antibodies including anti-Mouse alexa fluor 488, 594, anti-chicken alexa fluor 488, 647, and anti-rabbit alexa fluor 488, 594 (Molecular Probes, USA, 1 μg/ml dilution in PBS). Cryosections were washed three more times to remove secondary antibody before being mounted with Vectashield hardset mounting medium with nuclear stain (DAPI) following manufacturer’s instructions.

BrdU immunocytochemistry

BrdU is a thymidine analog that is incorporated into DNA during the S phase of the cell cycle and labels proliferating cells. To assess cell proliferation, control, and METH-treated human H9 derived NSC cultures and organoids were treated with BrdU (Sigma Aldrich B5002, 20 µM) for 12 h before fixing cells with 4% PFA and processing for BrdU immunocytochemistry. In brief, denature DNA of organoids sections/hNSCs with 2 N HCl for 10 min at 37 °C followed by neutralization with borate buffer (0.1 M, pH 8.5) for 10 min at room temperature. After blocking the cells for 30 min at room temperature, we used primary mouse antibodies for BrdU at 1:200 dilution in blocking buffer (BrdU, sc-32323, anti-mouse, 1:200) for overnight. The following day, wash and incubate cells with Alexa fluor 488 anti-mouse secondary labeling (1 μg/ml diluted in PBS) and counterstain with nuclear stain Hoechst 33342.

Immunoblotting and ELISA

Cerebral organoids (~50 mg) or astrocytes were lysed in 500 μl of Pierce IP lysis buffer containing protease inhibitor cocktail (Roche) and proteins were extracted by centrifugation at 13,000 × g for 10 min at 4 °C. The concentration of extracted proteins was determined by Bio-Rad DC protein assay kit as per manufacturer instructions (Bio-Rad) and equal amounts of protein (25 μg) were resolved by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% nonfat milk in PBST or Thermo Scientific Pierce Fast Western Blot Kits followed by overnight incubation at 4 °C with primary antibodies such as β-Actin (1:1000, Cell Signaling Technology (CST)-4970) and NLRP1 (1:1000, CST-4990). On following day, membranes were then washed three times with tris-buffered Saline with 0.1% Tween-20 (TBST) and incubated for 2 h with secondary antibody conjugated with horseradish peroxidase. Immunoreactive protein signals were detected with Super-signal West Pico Chemiluminescent Substrate (Pierce, USA). Densitometric analysis of protein bands were carried out by using Image Lab software (Bio-Rad, USA) and the values of proteins were normalized with β-Actin levels.

ELISA was performed using the Human IL-6 ELISA kit (eBioscience) following manufacturer’s instructions. Briefly, ELISA plates were incubated with IL-6 capture antibody overnight in coating buffer. Wells were washed with 0.05% PBST five times and blocked with Assay Diluent for 1 h at room temperature. After five additional washes, organoid supernatant collected at 4, 8, and 24 h post-METH treatment and IL-6 standards were added to wells and incubated overnight at 4 °C. The following day, wells were washed five times with PBST, incubated with diluted detection antibody for 1 h at room temperature. Wells were again washed five times before diluted Avidin-HRP was added for 30 min at room temperature. After seven washes, substrate solution was added for 15 min, followed by stop solution. ELISA plates were read by a Synergy 2 plate reader (Biotek) at 450 nm and 570 nm.

10x Chromium scRNA-seq

scRNA-seq was performed on eight untreated and six METH-treated organoids. Organoids were manually dissociated into single-cell suspension using the Milteny Biotec Neural Tissue Dissociation Kit (P) (Milteny Biotec, 130–092–628) following manufacturer’s instructions. Briefly, organoids were washed with PBS and cut into small pieces using a scalpel. Tissues were resuspended in warmed enzyme mix 1 containing papain and rotated for 15 min at 37 °C. Enzyme mix 2 was added and tissues were mechanically dissociated using a wide-tipped pipet. Cell suspensions were passed through a 40 μm cell strainer and centrifuged to pellet. Cell concentrations and viability were assessed by trypan blue staining.

scRNA-seq libraries were generated using the 10x Genomics Chromium Single-cell 3′ Library & Gel Bead Kit V2 (10x Genomics, PN-120237) at the UCSD Institute for Genomic Medicine Genomic Center. Libraries were generated from organoid single-cell suspensions following manufacturer’s protocol. Briefly, single-cell suspensions were mixed with RT primer and enzyme master mixes and loaded into the Single-cell 3′ Chip (10x Genomics, PN-120236) along with single-cell 3′ gel beads, and partitioning oil to generate GEMs using the Chromium Controller. GEMs were transferred and reverse transcribed to generate cDNA. After RT, cDNA was recovered, cleaned, and amplified. To construct the library, cDNAs were processed by fragmentation, end repair, and A-tailing. Libraries were size selected using SPRI-Select beads. P5 and P7 adapters were ligated and libraries were again size selected using SPRI-Select beads. Final libraries were analyzed using the Agilent TapeStation system and quantified using Qubit (Invitrogen). scRNA-seq libraries were sequenced using a HiSeq4000.

Bioinformatic analysis

Chromium single-cell 3′ RNA-seq libraries were aligned to GRCh38.p11 using the 10x Genomics Cell Ranger 2.0.0 pipeline following manufacturer’s default settings. Briefly, alignment, filtering, and UMI counting was performed using cell ranger count (10x Genomics) and aggregated using cell ranger aggr. Tertiary analyses on cell ranger output files were performed using Seurat [48]. Data normalization, filtering, clustering, and differential gene expression analyses were performed using Seurat R package version 2.4 and 3.0 with R version 3.5.3 [25, 48]. Integrative single-cell analyses were performed following previously established Seurat pipelines with modifications [25]. Cells with fewer than 200 genes were filtered out. To integrate control and METH-treated samples, datasets were aligned using highly variable genes within samples with the Run Multi CCA and Align Subspace functions (Seurat V2.4). TSNE clustering was used to determine the clusters of the cells. Differential gene expression analyses were performed by subsetting the data based on cluster identity and assuming a negative-binomial distribution. Treatment response to determine up and down differentially expressed genes due to METH treatment was determined by finding the markers between all of the cells between the two treatment conditions outlined in the immune alignment tutorial for Seurat. The list of variable genes was separated into two separate gene lists based on up and down regulation. The entire list of expressed genes was used to create a reference gene list for GO analysis via PANTHER for overrepresentation analysis (http://geneontology.org/). The up and downregulated genes were uploaded into PANTHER separately and the results from the two analyses were sorted based on raw p value. The log of the raw p value of these results was calculated and the top 9 downregulated pathways and top 20 upregulated pathways were used with the ggplot2 package version 3.1.1 in R to create Fig. S2. All dot plots and violin plots were created using functions from the Seurat package version 3.0.