Antisense oligonucleotide therapeutic approach for Timothy syndrome – Nature

Culture of hiPS and HEK293T cells

The hiPS cells in this study were previously described and validated^2,3. A total of six hiPS cell lines were derived from fibroblasts collected from three healthy individuals and three with TS. Approval for this study was obtained from the Stanford IRB panel, and informed consent was obtained from all participants. The isogenic TS1 (G406R) line was derived in the KOLF2.1 hiPS cell line via nucleofection using the guide RNA-targeting GGTGTGCTTAGCGG and the homologous repair template ssODN, aggaatagcagaaagaataaataaaaataaatggaaaaatcaagacctttttccttggtcctgcttacCTGCTAAGCACACCGAGAACCAAGTTAAGTAC³³. The CW30293 hiPS cell line was obtained from CIRM. The presence of the heterozygous mutation was confirmed by Sanger sequencing. hiPS cells were cultured in feeder-free essential 8 medium (E8, Thermo Fisher Scientific, catalogue no. A1517001) without antibiotics and kept in the wells of six-well plates (Corning, catalogue no. 3506) coated for 1 h at room temperature with vitronectin recombinant human protein (VTN-N, Thermo Fisher Scientific, no. A14700) diluted 1:100 to a final concentration of 5 ng ml⁻¹ in Dulbecco’s PBS (DPBS), with neither calcium nor magnesium (Thermo Fisher Scientific, catalogue no. 14190136). To facilitate passaging, hiPS cells were first washed with DPBS and then incubated with 0.5 mM EDTA (Invitrogen, catalogue no. 15575020) in DPBS at room temperature for 7 min. Following removal of EDTA solution, cells were seeded in fresh wells of six-well plates coated with VTN-N and containing E8 medium. The hiPS cells used in this study were maintained free of Mycoplasma at 37 °C in a humidified-air atmosphere with 5% CO₂. The lenti-X 293T cell line, a subclone of HEK293T cells, was obtained from Takara Bio (catalogue no. 632180) and cultured in DMEM (Gibco, catalogue no. 10313021) supplemented with 10% fetal bovine serum (Corning, catalogue no. 35016CV) and 1× GlutaMAX (Thermo Fisher Scientific, catalogue no. 35050061). This cell line was chosen because it is compatible with robust plasmid overexpression.

Generation of hCO and hSO from hiPS cells

The generation of hCO, hSO and hFA was performed as previously described^3,34,35. In brief, hiPS cells were incubated with Accutase (Innovate Cell Technologies, no. AT-104) at 37 °C for 7–8 min and dissociated into single hiPS cells. Single-cell suspensions were collected in a 50 ml Falcon tube and cell pellets obtained via centrifugation at 300g for 3 min. Cell numbers were counted following resuspension of cell pellets. Approximately 3 × 10⁶ cells in 2 ml of E8 medium supplemented with ROCK inhibitor Y-27632 (10 μM, Selleckchem, catalogue no. S1049) were added per well of an AggreWell 800 plate (STEMCELL Technologies, catalogue no. 34815). The plates were then centrifuged at 100g for 3 min to allow cells to sink to the bottom of the wells (day 0). Twenty-four hours following cell aggregation (day 1), spheroids were dislodged by pipetting (with a P1000 tip cut at the end) and transferred to ultralow-attachment plastic dishes (Corning, no. 3262) in essential 6 medium (E6, Life Technologies, no. A1516401) supplemented with 2.5 μM dorsomorphin (Sigma-Aldrich, catalogue no. P5499) and 10 μM SB-431542 (Tocris, catalogue no. 1614). From days 2 to 6, E6 medium was changed daily and supplemented with dorsomorphin and SB-431542. In addition the Wnt pathway inhibitor XAV-939 (XAV, 1.25 μM, Tocris, catalogue no. 3748) was added, together with dorsomorphin and SB-431542. On the seventh day in suspension, basal medium was switched to neural medium consisting of Neurobasal A (Life Technologies, catalogue no. 10888), B-27 supplement without vitamin A (B-27, Life Technologies, catalogue no. 12587), GlutaMAX (1:100, Life Technologies, catalogue no. 35050) and 10 U ml⁻¹ penicillin-streptomycin (Gibco, catalogue no. 15140122). From days 6 to 24 the neural medium was supplemented with 20 ng ml⁻¹ epidermal growth factor (EGF, R&D Systems, catalogue no. 236-EG) and 20 ng ml⁻¹ basic fibroblast growth factor (FGF, R&D Systems, catalogue no. 233-FB) for 19 days (until day 24), with medium changed daily from days 7–18 and every other day until day 24. From days 25–42 the neural medium contained 20 ng ml⁻¹ brain-derived neurotrophic factor (Peprotech, catalogue no. 450-02) and 20 ng ml⁻¹ NT3 (Peprotech, catalogue no. 450-03), with medium change every other day. From day 43, hCO were cultured with only neural medium without growth factors. The generation of hSO differs from that of hCO in that, from days 7–12, the neural medium was supplemented with XAV (1.25 μM) in addition to EGF and FGF; from days 13–24 the neural medium was supplemented with XAV (1.25 μM) and SAG (100 nM, EMD Millipore, catalogue no. 566660) in addition to EGF and FGF.

ASOs

ASOs were 20-nt-long synthesized using the phosphorothioate backbone and with a MOE modification. 5-Methylcytosine was used during synthesis rather than cytosine. ASOs tested on hiPS cell-derived forebrain organoids were purified by standard desalting followed by Na⁺ salt exchange. These ASOs were reconstituted in nuclease-free water at a concentration of 1 mM and stored at −20 °C thereafter for in vitro experiments. For in vivo injection, ASO.14 was reconstituted at a concentration of 10 μg μl⁻¹ in DPBS for injection of 30 μl of 300 μg ASO into rat cisterna magna. All ASOs used in this study were manufactured by Integrated DNA Technologies. Cy5-labelled ASOs were synthesized by the addition of Cy5 to the 5′ of the ASO (Integrated DNA Technologies) followed by HPLC purification and Na⁺ salt exchange.

Recombinant DNA and viruses

pDup4-1 was obtained from Addgene (plasmid no. 23022) and was used as the backbone for the minigene splicing reporter. pDup4-1 was digested with ApaI and BglII (New England Biolabs) and the resulting 4,595 bp fragment was purified following loading on a 1% agarose gel using the QIAquick PCR Purification Kit (Qiagen, catalogue no. 28106). Genomic DNA from TS hiPS cells was purified with the DNeasy Blood & Tissue Kit (Qiagen, catalogue no. 69506). Amplicons encompassing exons 8 and 8A of CACNA1C were amplified with GoTaq Long PCR Master Mix (Promega, catalogue no. M4021). Primer sequences and cycling conditions are listed in Supplementary Tables 1 and 2. Purified PCR products were digested with ApaI and BglII. Following one further round of purification, DNA was dephosphorylated with FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, catalogue no. EF0654) then ligated to the pDup4-1 backbone using T4 DNA ligase (Thermo Fisher Scientific, catalogue no. EL0011). Following transformation (One Shot Stbl3 Chemically Competent E. coli, Thermo Fisher Scientific, catalogue no. C737303), colonies were picked for sequence verification. The human PTBP1 ORF plasmid was obtained from Genscript (clone ID OHu15891D, accession no. NM_002819.5). Plasmids encoding WT and TS Ca_V1.2 were synthesized by VectorBuilder based on transcript ENST00000399655.6 under a CAG promoter into a lentivirus backbone. An HA tag was placed in between Q683 and T684. The GCaMP plasmid was obtained from Addgene (plasmid no. 111543). Plasmids encoding the β1b and a2δ subunits of the L-type calcium channel were described previously⁵. The maps and sequences of minigene splicing reporters and human Ca_V1.2 expression plasmids are included in Supplementary Figs. 3–6 (generated by SnapGene 5.1.4.1, SnapGene software from Dotmatics).

RNA extraction and qPCR

For all samples, RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, catalogue no. 74136). Unless otherwise noted, reverse transcription was performed using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, catalogue no. 11752050) according to the manufacturer’s instructions. qPCR was performed on a QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific, catalogue no. 4485689) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, catalogue no. 4312704). Primers for qPCR are listed in Supplementary Tables 1 and 2.

Transcript analysis of CACNA1C exons 8 and 8A

Restriction fragment-length polymorphism analysis of CACNA1C exons 8 and 8A was performed on PCR fragments amplified from cDNA. DNA was purified using AMPure XP beads (Beckman Coulter, catalogue no. A63881) according to the manufacturer’s instructions. Purified DNA was digested with BamHI (Thermo Fisher Scientific, catalogue no.ER0055) at 37 °C for 3 h and loaded on 2% agarose gel. Gel images were taken on a Gel Doc XR+ imager (Bio-Rad, catalogue no. 1708195). For next-generation sequencing analysis of transcripts, primers with the Illumina adaptor were used to amplify the region encompassing exons 7–9. Following bead purification, DNA was eluted in water and sent for sequencing using the Genewiz Amplicon-EZ module. Next-generation sequencing analysis of the minigene splicing reporter was performed similarly by amplifying minigene transcripts from the cDNA of transfected HEK cells 3 days post transfection. Primers and cycling conditions are listed in Supplementary Tables 1 and 2.

Transfection of HEK cells

Approximately 30,000–75,000 HEK cells were seeded per well of a 24-well plate (Corning, catalogue no. 353047). The following day, plasmids were mixed with 1 mg ml⁻¹ PEI MAX (Polysciences, catalogue no. 24765-1) in 50 μl of a 150 mM NaCl solution. Following about 10 s of vigorous vortexing, plasmid mixtures were incubated for 15 min at room temperature and then added to the wells (Supplementary Tables 3–5).

Dissociation for monolayer culture

Dissociation of hCO for monolayer culture was performed as previously described, with minor optimizations⁴. Coverslips were coated with approximately 0.001875% polyethylenimine (PEI, Sigma-Aldrich, catalogue no. 03880) for 1 h at 37 °C, washed four times with water and dried. On the day of dissociation, betweeen four and six hCO per hiPS cell line were transferred to wells in six-well plates (Corning, catalogue no. 3506) and incubated for 45–60 min at 37 °C with 3 ml of enzymatic dissociation solution. This solution consisted of 30 U ml⁻¹ papain (Worthington Biochemical, catalogue no. LS003127), 1× EBSS (Millipore Sigma, catalogue no. E7150), 0.46% d(+)-glucose, 0.5 mM EDTA, 26 mM NaHCO₃, 10 μM Y-27632, 125 U ml⁻¹ deoxyribonuclease I (Worthington Biochemical, catalogue no. LS002007) and 6.1 mM l-cysteine (Millipore Sigma, catalogue no. C7880). Following papain incubation, samples were collected in a 15 ml Falcon tube and centrifuged at 1,200 rpm for 1 min. Following removal of the supernatant, samples were washed with 1 ml of inhibitor solution with 2% trypsin inhibitor (Worthington Biochemical, catalogue no.LS00308) and resuspended in 1 ml of the same solution for trituration. Following trituration, 1 ml of inhibitor solution with 4% trypsin inhibitor was added slowly beneath the cell suspension to create a gradient layer; the gradient solution was then centrifuged at 1,200 rpm for 5 min. Cell pellets were resuspended in culture medium consisting of Neurobasal A supplemented with B-27 and 10 μM Y-27632. Undissociated tissue was removed by passing the cell suspension through a 40 μm cell strainer (Corning, catalogue no. 352340). Finally, dissociated cells were seeded on the coverslip at a density of 50,000 cells per coverslip in 1 ml of culture medium. The inhibitor solution differs from the enzyme solution in that it contains neither papain nor EDTA. All centrifugation steps were performed at room temperature.

Calcium imaging

Fura-2 calcium imaging on monolayer hCO cells was performed as previously described²⁶. In brief, cells were loaded with 1 mM Fura-2 acetoxymethyl ester (Fura-2 AM, Invitrogen, no. F1221) for 30 min at 37 °C in NM medium, washed with NM medium for 5 min and then transferred to a perfusion chamber (RC-20, Warner instruments) in low-potassium Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose, 25 mM HEPES pH 7.4) on the stage of an inverted fluorescence microscope (Eclipse TE2000U, Nikon). Following 0.5 min of baseline imaging, high-potassium Tyrode’s solution was perfused for 1 min. Imaging was performed at room temperature (25 °C) on an epifluorescence microscope equipped with an excitation filter wheel and an automated stage. Openlab software (PerkinElmer) and IGOR Pro (v.5.1, WaveMetrics) were used to collect and quantify time-lapse excitation 340:380-nm-ratio images at an imaging rate of approximately 1 Hz, as previously described²⁰. Residual calcium was calculated as (C − A)/(B − A), where A is the baseline value (fifth frame), B is the peak value following depolarization (manually determined) and C is the decay value (B + 25th frame).

For GCaMP imaging, HEK293T cells were seeded in 24-well plates. The following day, cells were transfected with a mixture of plasmids including subunits Ca_V1.2 β1b, α2δ and α1 and GCaMP6-X (Supplementary Table 3). Three days post transfection, imaging was performed with an SP8 confocal microscope (Leica Microsystems) at a frame interval of 1.2875 s. Before imaging, cell culture medium was replaced with 500 μl of 5 mM Tyrode’s solution. Following 30 s of baseline imaging, 500 μl of 129 mM Tyrode’s solution (final concentration 67 mM KCl) was added.

Similarly, for GCaMP imaging in two-dimensional neurons, TS and WT hCO were dissociated into 24-well imaging plates (Cellvis P24-0-N) and infected with AAV-DJ-hSYN1::GCaMP6f (Gene Vector and Virus Core, Wu Tsai Neurosciences Institute, Stanford University). Various concentrations of ASOs (ASO.14, ASO.17, ASO.18 and ASO.Scr) were applied to dissociated neurons. After 10 days, GCaMP imaging was carried out with an SP8 confocal microscope using the 20× objective at 1.2875 s per frame). Before imaging, culture medium was replaced with 500 μl of 5 mM Tyrode’s solution. Following 30 s of baseline imaging, 500 μl of 129 mM Tyrode’s solution (final concentration 67 mM KCl) was added. Imaging was acquired over a total time of 8 min.

For GCaMP imaging analysis of HEK293T cells, regions of interest (ROIs) corresponding to cell somas were identified semiautomatically using a custom-written ImageJ segmentation macro. ROIs were detected in the frame following depolarization (fifth or sixth frame following KCl administration) by applying a mask, watershedding and using the ‘Analyze particles’ function (size 10–1,000, circularity 0.4–1.0). A minority of ROIs were manually excluded due to either cell drift, off-target detection of background or detection of more than a single soma within the same ROI. For GCaMP analysis in neurons, ROIs corresponding to cell somas were manually annotated. Downstream analyses for both HEK293T cells and neurons were performed using custom-written R codes. Mean grey values were transformed to relative changes in fluorescence: dF/F(t) = (F(t) − F₀)/F₀, where F₀ represents average grey values of the time series of each ROI. Cells were excluded if their amplitude was lower than the baseline mean or more than 20× baseline mean. Residual calcium values were calculated as described above, with B representing peak value, A baseline value (20 frames upstream of the peak-value frame) and C decay value (200 frames after the peak-value frame). Extreme residual calcium values (lower than −5 or higher than +5) were excluded.

Patch-clamp recordings

Patch-clamp recordings were performed on cortical neurons dissociated from hCO, as previously described⁴. hCO were dissociated at days 100–150. A few days following dissociation, cells were infected with AAV-DJ-SYN1::eYFP and 1 μM ASO was added 1 week following dissociation. Recordings were typically made around 3–4 weeks following dissociation. Cells were identified as eYFP⁺ with an upright slice scope microscope (Scientifica) equipped with an Infinity2 CCD camera and Infinity Capture software (Teledyne Lumenera). Recordings were performed with borosilicate glass electrodes with a resistance of 7–10 MΩ. For barium current recordings the external solution contained 100 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 20 mM BaCl₂, 25 mM TEA-Cl, 4 mM 4-AP, 10 mM HEPES and 20 mM glucose pH 7.4, with NaOH and 300 mOsm. The internal solution contained 110 mM CsMethylSO₃, 30 mM TEA-Cl, 10 mM EGTA, 4 mM MgATP, 0.3 mM Na₂GTP, 10 mM HEPES and 5 mM QX314-Cl pH 7.2, with CsOH and 290 mOsm. Data were acquired with a MultiClamp 700B Amplifier (Molecular Devices) and a Digidata 1550B Digitizer (Molecular Devices), low-pass filtered at 2 kHz, digitized at 20 kHz and analysed with pCLAMP (v.10.6, Molecular Devices). Cells were subjected to −10 mV hyperpolarization (100 ms) every 10 s to monitor input and access resistance. Cells were excluded for analysis if they showed a change of over 30%. Liquid junction potential was not corrected in this study.

For barium current recordings, cells were recorded in the presence of tetrodotoxin (TTX) (0.5 μM) to block sodium currents and were held at −70 mV in voltage-clamp and depolarizing voltage steps (5 s for the majority of cells, from −70 to +20 mV) in increments of 5 mV. Inactivation of barium current was calculated from cells subjected to 5 s or 2–3-s depolarization steps at 2 s under maximal current (−20 to 0 mV for the majority). For some cells, recordings with a prestep of −110 mV (or −100 mV) hyperpolarization were also included for inactivation at 2 s. Leak subtraction was used to minimize the artefact of membrane resistance in MultiClamp 700B. I–V curves were fitted in Origin (OriginPro 2021b, OriginLab) with a Boltzmann exponential function: I = G_max × (V − E_Ba)/{1 + exp[(V_0.5 − V)/K]}, where G_max is the maximal conductance of calcium channels, E_Ba is the reversal potential of barium estimated by the curve-fitting programme, V_0.5 is the potential for half-maximal, steady-state activation of barium current and K is a voltage-dependent slope factor.

For voltage-dependent barium current inactivation, cells were held at −70 mV. A series of prepulse voltage steps (3 s) were administered, from −110 or −100 to +40 mV, in increments of 10 mV. Testing of the voltage step (−10 or 0 mV, where maximal current was recorded) was then carried out for a further 1–3 s. Barium current inactivation was calculated as relative current normalized to current amplitude from the first test pulse. Voltage-dependent inactivation curves were fitted with exponential functions in Origin.

Immunostaining

Dissociated cells from TS hCO at 100–120 days of differentiation were plated on precoated coverslips and placed in wells of a 12-well plate; different concentrations of Cy5-ASO.14 were then added. After 3 days the coverslips were first fixed for 10 min at room temperature with a solution containing one volume each of culture medium and fixation buffer comprising 4% paraformaldehyde (PFA) and 4% sucrose in DPBS. Next, two volumes of fixation buffer were added for an extra 20 min to finalize the fixation step. Following two rounds of washing with DPBS, coverslips were incubated for 1 h with blocking buffer consisting of 0.3% Triton X-100 and 10% normal donkey serum prepared with PBS. Following removal of the blocking buffer, primary antibodies were added for overnight incubation at 4 °C. Antibodies CTIP2 (abcam, catalogue no. ab18465) and SATB2 (abcam, catalogue no. ab51502) were diluted in blocking buffer at 1:300. Coverslips were washed twice with DPBS then incubated with secondary antibody (1:1,000 in blocking buffer; donkey anti-rat Alexa 488, Thermo Fisher Scientific, catalogue no. A-21208; and donkey anti-mouse Alexa 568, Thermo Fisher Scientific, catalogue no. A10037) at room temperature for 1 h. Following a further two rounds of washing with DPBS, Hoechst 33258 (Thermo Fisher Scientific, catalogue no. H3569) was added to coverslips for 10 min followed by a final round of washing with DPBS. Finally, coverslips were mounted on slides (Fisherbrand Superfrost Plus Microscope Slides, Fisher Scientific, catalogue no. 12-550-15) using Aqua-Poly/Mount (Polysciences, catalogue no. 18606). Images were acquired with a confocal SP8 (Leica Microsystems) using a 20× objective.

The TUNEL assay was performed using the in situ cell death detection kit (Roche, catalogue no. 12156792910). In brief, hCO were dissociated and exposed to either 1 μM ASO or scrambled control for 48 h. Cells were then fixed in 4% PFA, permeabilized in Triton X-100 and incubated with TUNEL reaction solution for 1 h at 37 °C. Samples pretreated with DNase1 for 10 min were used as positive control. Following rinsing and counterstaining with Hoechst, coverslips were imaged with a Stellaris microscope using the 20× objective. Images were stitched in Fiji and a custom macro was used to split channels, set thresholds for detection of nuclei via Hoechst and determine Cy3⁺ nuclei via thresholds set blindly on control samples.

For c-Cas3, immunostaining was performed as for Cy5 samples except that rabbit anti-c-Cas3 (Asp175) (1:300, CST, catalogue no. 9661S) and mouse anti-MAP2 antibody (1:100, Sigma-Aldrich, catalogue no. M4403) were used as primary antibodies and donkey anti-rabbit 568 (1:1,000, Thermo Fisher Scientific, catalogue no. A10042) and donkey anti-mouse Alexa:568 (1:1,000, Thermofisher Scientific, catalogue no. A10037) as secondary antibodies. Coverslips were imaged with a confocal SP8 microscope using the 40× objective. Three to four fields were acquired per coverslip. Images were analysed using Fiji with maximal projection, standardized thresholding and circularization to identify cells (via Hoechst nuclear staining) and then c-Cas3⁺ cells (via Cas3 staining).

For staining of t-hCO, following slicing of fresh rat brain containing t-hCO, slices were postfixed in 4% PFA overnight at 4 °C and then washed three times with PBS. Next, slices were incubated with blocking buffer at room temperature for 1 h with 10% normal donkey serum and 0.3% (vol/vol) Triton X-100 in DPBS then incubated with primary antibody diluted in blocking buffer overnight at 4 °C (anti-HNA, mouse, 1:200, abcam, catalogue no. ab191181). Washing steps, staining with secondary antibody and staining of nuclei are described above.

Flow cytometry

TS hCO were incubated with 1 μM Cy5.ASO.14 in wells of 24-well, ultralow-attachment plate (Corning, catalogue no. 3473) for 2 days. hCO were then dissociated and resuspended in 200 μl of staining buffer containing 3% bovine serum albumin and 0.5 mM EDTA. Cells were incubated either with or without PE Mouse Anti-Human CD90 (BD Biosciences, catalogue no. 555596, dilution 1:100) for 30 min at 4 °C. Next, three rounds of washing steps were performed using the staining buffer and cells were resuspended in 200 μl of staining buffer and passed through a 40 μm cell strainer. Non-treated hCO not stained with CD90 served as a control for setting up the gate during cell acquisition. G575 and R670 were used for measurement of PE and Cy5 signal, respectively. Flow cytometry was performed on a BD Aria cell sorter at the Stanford Shared FACS Facility according to the Facility’s calibration instructions. Data were processed using FlowJo 10.7.1 software (BD).

Immunoblot for measurement of Ca_V1.2 protein level

hCO derived from control and TS iPS cell lines were aliquoted to wells of a 24-well, ultralow-attachment plate (Corning, catalogue no. 3473). Each well contained two or three hCO cultured in 2 ml of neural medium, followed by the addition of 1 μM ASO. Medium was 50% replaced following 3 days of ASO exposure and samples collected following 7 days of ASO exposure. Protein lysates for hCO were prepared using the RIPA buffer system (Santa Cruz, catalogue no. sc-24948). Protein lysates of t-hCO were prepared by the brief addition of 50 µl of SDS Buffer (1.5% SDS, 25 mM Tris pH 7.5) in a 1.5 ml tube followed by sonication (Qsonica Q500 sonicator; pulse 3 s on, 3 s off, amplitude 20%). Protein concentrations were quantified using the bicinchoninic acid assay (Pierce, ThermoFisher, catalogue no. 23225): 20 μg of protein per sample per lane was loaded and run on a 4–12% Bis-Tris PAGE gel (Bolt 4–12% Bis-Tris Protein Gel, Invitrogen, no. NW04122BOX) and transferred to a polyvinylidene difluoride membrane (Immobulin-FL, EMD Millipore, catalogue no. IPFL00010). Membranes were blocked with 5% bovine serum albumin in Tris buffered saline with Tween (TBS-T) for 1 h at room temperature and incubated overnight with primary antibodies against GAPDH (mouse, 1:5,000, GeneTex, catalogue no. GTX627408) and Ca_V1.2 (rabbit, 1:1,000, Alamone labs, catalogue no. ACC-003) for 48 h for hCO samples, and for 96 h for transplanted samples, at 4 °C. Membranes were washed three times with TBS-T and then incubated with near-infrared fluorophore-conjugated species-specific secondary antibodies—either goat anti-mouse IgG polyclonal antibody (IRDye 680RD, 1:10,000, LI-COR Biosciences, catalogue no. 926–68070) or goat anti-rabbit IgG polyclonal antibody (IRDye 800CW, 1:10,000, LI-COR Biosciences, catalogue no. 926–32211), for 1 h at room temperature. Following the application of secondary antibody, membranes were washed three times with TBS-T, once with TBS and then imaged using a LI-COR Odyssey CLx imaging system (LI-COR).

TLR9 assay for ASO toxicity

We used the human TLR9 reporter assay (Invivogen, catalogue no. hkb-htlr9) according to the manufacturer’s instructions. In brief, modified HEK293T cells were grown on 100 mm cell culture plates to 50–80% confluency. They were then detached in PBS, resuspended at 450,000 cells ml⁻¹ in HEKBlue solution and replated into a 96-well plate. Positive controls were exposed to ODN2006 (Invivogen, catalogue no. tlrl-2006), and negative controls to sterile water; other samples were exposed to 1 μM ASO for 16–24 h. Following exposure, TLR9 activation was detected by spectrophotometer (620–655 nm absorption) using a monochromator plate reader (Tecan, Infinite M1000) and XFluor 2.0 software.

Interneuron migration and imaging analysis

Following 45–50 days of differentiation, hSO were incubated overnight with LV.Dlxi1/2b::eGFP lentiviral particles in an Eppendorf tube and transferred to a 24-well plate. After 3–7 days, hSO were coincubated with an hCO in an Eppendorf tube supplemented with 1 ml of medium to generate hFA, which were then cultured in a single well of an ultralow-attachment 24-well plate (Corning). Baseline imaging of interneuron migration was taken around 3–4 weeks following the formation of hFA. Next, 1 μM ASO was added to hFA followed by reimaging 2 weeks later. All imaging was taken over a period of 20 min for 12–15 h inside a confocal chamber at 37 °C in a humidified-air atmosphere with 5% CO₂. Quantification of saltation length and frequency was performed as previously described³. Only mobile cells were included for analysis. ImageJ was used for analysis of interneuron migration. In cases where hFA moved during imaging, linear stack alignment with SIFT was used to correct minor shifts. To estimate the distance of individual saltations, Dlxi1/2b::eGFP cells showing a swelling of the soma were identified and distance (in μm) to the new position of the soma following nucleokinesis was recorded manually. The time necessary for this movement was used to calculate the speed when mobile. Typically, only cells showing two or more saltation movements were included.

Transplantation into athymic newborn rats

Animal procedures were performed following animal care guidelines approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC). Pregnant RNU euthymic (rnu/⁺) rats were either purchased (Charles River Laboratories) or bred in house. Animals were maintained under a 12/12 h light/dark cycle and provided food and water ad libitum. Three-to-seven-day-old athymic (FOXN1^−/−) rat pups were identified by immature whisker growth before culling. Pups (both male and female) were anaesthetized with 2–3% isoflurane and mounted on a stereotaxic frame. A craniotomy, of about 2–3 mm in diameter, was performed above S1, preserving the dura intact. Next, the dura mater was punctured using a 30-G needle (approximately 0.3 mm) close to the lateral side of the craniotomy. A hCO was next moved onto a thin, 3 × 3 cm parafilm and excess medium removed. Using a Hamilton syringe connected to a 23-G, 45° needle, the hCO was gently pulled into the distal tip of the needle. The syringe was next mounted on a syringe pump connected to the stereotaxic device. The sharp tip of the needle was positioned above the 0.3-mm-wide prefabricated puncture in the dura mater (z = 0 mm) and the syringe was reduced by 1–2 mm (z = approximately −1.5 mm) until a tight seal between needle and dura mater had formed. Next, the syringe was elevated to the centre of the cortical surface at z = −0.5 mm and the hCO ejected at a speed of 1–2 μl min⁻¹. Following completion of hCo injection, the needle was retracted at a rate of 0.2–0.5 mm min⁻¹, the skin was closed and the pup immediately placed on a warm heat pad until complete recovery.

MRI of transplanted rats

All animal procedures followed animal care guidelines approved by Stanford University’s APLAC. Rats (more than 60 days post transplantation) were anaesthetized with 5% isoflurane for induction and 1–3% isoflurane during imaging. For imaging, an actively shielded Bruker 7 Tesla horizontal bore scanner (Bruker Corp.) with International Electric Company gradient drivers, a 120-mm-inner-diameter shielded gradient insert (600 mT m⁻¹, 1,000 T m⁻¹ s⁻¹), AVANCE III electronics, eight-channel multicoil radiofrequency and multinuclear capabilities, and the supporting Paravision 6.0.1 platform, were used. Acquisitions were performed with an 86-mm-inner-diameter actively decouplable volume radiofrequency coil with a four-channel, cryocooled, receive-only radiofrequency coil. Axial two-dimensional Turbo-RARE (TR 2,500 ms, TE 33 ms, two averages) 16-slice acquisitions were performed at 0.6–0.8 mm slice thickness with samples of approximately 256 Å. Signal was received by a 2-cm-inner-diameter quadrature transmit–receive volume radiofrequency coil (Rapid MR International). Successful transplantations were defined as those resulting in a continuous area of T2-weighted MRI signal in the transplanted hemisphere.

ASO injection into rat cisterna magna

Rats were anaesthetized with 5% isoflurane for induction and 2–3% isoflurane during ASO injection through the cisterna magna. Animals were placed in the prone position with a small paper roll under the neck to tilt the head downwards. The neck was shaved and wiped clean with ethanol. To target the cisterna magna the foramen magnum was determined by touch and a 27-G needle attached to a syringe (BD, catalogue no. 305620) filled with 300 μg of ASO was percutaneously inserted into the cisterna magna perpendicularly to the neck. The needle was held with the bevel face upwards and 30 μl of ASO was slowly injected into the cisterna magna. The procedure took less than 2 min per rat. Animals recovered from anaesthesia within 10 min of isoflurane induction. ASO injections were performed in rats with t-hCO at 162–258 days and were not blinded. Sample sizes were estimated empirically.

Processing of ASO-injected rats

Rats were anaesthetized with isoflurane and brain tissue was removed and placed in cold (approximately 4 °C), oxygenated (95% O₂ and 5% CO₂) sucrose slicing solution containing 234 mM sucrose, 11 mM glucose, 26 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM MgSO₄ and 0.5 mM CaCl₂ (approximately 310 mOsm). Coronal rat brain slices (300–400 μm) containing t-hCO were sectioned using a Leica VT1200 vibratome as previously described³. t-hCO sections were then moved to a continuously oxygenated slice chamber, at room temperature, which contained aCSF (10 mM glucose, 26 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaHPO₄, 1 mM MgSO₄, 2 mM CaCl₂ and 126 mM NaCl (298 mOsm)).

Calcium imaging in t-hCO from rats receiving ASO injection

Following dissection and sectioning of rat brains with t-hCO, slices were incubated with Calbryte 520 AM (AAT Bioquest, catalogue no. 20650) in 1:1 of NPC medium and PBS for 45–60 min at 37 °C. Slices were then transferred to a 24-well imaging plate containing 500 μl of warm, low-potassium Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose, 25 mM HEPES pH 7.4) and imaged with a confocal microscope (Leica Stellaris) for 30 s at 37 °C, after which medium was replaced by high-potassium Tyrode’s solution (high-KCl, 67 mM KCl: 67 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose and 25 mM HEPES pH 7.4) and imaging resumed for 25 min. Mean grey values were collected from ROIs delineating Calbryte⁺ somas (visualized by standard deviation projection of the entire time series) with Fiji (ImageJ v.2.1.0, NIH). Mean grey values were transformed to relative changes in fluorescence: dF/F(t) = (F(t) − F₀)/F₀, where F₀ represents average grey values of the time series of each ROI. Residual calcium was calculated as (C − A)/(B − A), where B is the peak value following depolarization (maximal peak value determined by custom-written MATLAB routines (v. R2019b and v. R2022b, 9.4.0, MathWorks), A is the baseline value (B − 50th frame) and C is the decay value (B + 150th frame).

Golgi staining

Golgi staining was conducted using the FD Rapid GolgiStain Kit (FD Neurotechnologies, catalogue no. PK401) according to the manufacturer’s instructions. In brief, freshly dissected t-hCO were incubated with solution A/B mixture in the dark and then transferred to solution C. After 72 h the tissue was embedded in agarose, the vibratome chamber filled with solution C and tissue sectioned at 100 μm using a Leica VT1200S vibratome. Sections were mounted on gelatin-coated slides, stained in solution D/E, washed, dehydrated, cleared and coverslipped. Images were acquired on a SP8 confocal microscope with brightfield. Cells were counted as neurons based on their morphology; dendrites were manually traced using neuTube. Both tracing and analysis were performed blinded.

Statistics and reproducibility

Data are presented as either mean ± s.d. or mean ± s.e.m. unless otherwise indicated. Distribution of raw data was tested for normality of distribution; statistical analyses were performed using either two-tailed student’s t-tests, one-way ANOVA with multiple comparisons, two-tailed Mann–Whitney tests or Kruskal–Wallis tests. Statistical analysis was performed in Prism (GraphPad). Data shown for representative experiments were repeated, with similar results, in at least three independent biological replicates, unless otherwise noted. Sample sizes were estimated empirically.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.