GoKawiilGenetically encoded cortical depletion
Emx1-cre (JAX, B6.129S2-Emx1tm1(cre)Krj/J), Esco2fl/fl (JAX, B6N.129S-Esco2tm1.1Ge/J) and C57BL SCID (JAX, B6.Cg-Prkdcscid/SzJ) mice were used to generate the breeding scheme: male Esco2fl/flPrkdcscid/scid × female Emx1-cre+/−Esco2fl/+Prkdcscid/scid. Breeder and offspring cages were supplemented with breeder chow (Envigo Teklad 2919) and DietGel 76A (with plant protein) to help to increase pup survival. Some breeders were more prone to parental infanticide. In these cases, we implemented an aunting strategy with Swiss Webster active dams from Charles River. To reduce competition for maternal care and milk, a subset of pups was euthanized during the second postnatal week.
hiPS cells, generation of hCOs and viral infection
We generated and cultured hCOs from hiPS cells as previously described53,54. In brief, hiPS cells were treated with Accutase (Innovate Cell Technologies, AT-104) at 37 °C for 7 min to dissociate them into single cells. The resulting single-cell suspension was collected in a 50-ml Falcon tube, and a cell pellet was obtained by centrifugation at 200g for 4 min. After resuspending the cell pellets, cell counting was performed. Approximately 3 × 106 cells in Essential 8 medium (Life Technologies, A1517001) supplemented with the ROCK inhibitor Y-27632 (10 μM; Selleckchem, S1049) were added to each well of the AggreWell 800 plate (StemCell Technologies, 34815). The plates were then centrifuged at 100g for 3 min to capture the cells in the microwells and incubated at 37 °C with 5% CO 2 (day −1). Then, 24 h after cell aggregation (day 0), spheroids were collected from each microwell by gently pipetting the medium up and down using a cut P1000 tip and transferring them into ultra-low attachment plastic dishes (Corning, 3262) in Essential 6 medium (Life Technologies, A1516401) supplemented with dorsomorphin (2.5 μM; Sigma-Aldrich, P5499) and SB-431542 (10 μM; Tocris, 1614). From days 2 to 5, the Essential 6 medium was changed daily and supplemented with dorsomorphin and SB-431542. On the sixth day in suspension, the neural spheroids were transferred to neural medium composed of Neurobasal A (Life Technologies, 10888), B-27 supplement without vitamin A (Life Technologies, 12587), GlutaMax (1:100, Life Technologies, 35050) and 10 U ml−1 penicillin–streptomycin (Gibco, 15140122). From days 6 to 24, the neural medium was supplemented with 20 ng ml−1 epidermal growth factor (EGF; R&D Systems, 236-EG) and 20 ng ml−1 basic fibroblast growth factor (FGF; R&D Systems, 233-FB), with medium changes occurring daily from days 6 to 15 and every other day until day 24. From days 25 to 42, the neural medium included 20 ng ml−1 brain-derived neurotrophic factor (BDNF; Peprotech, 450-02) and 20 ng ml−1 NT-3 (Peprotech, 450-03), with medium changes every other day. Starting from day 43, the hCOs were cultured in neural medium without growth factors, with medium changes every 4 days.
For viral labelling, all organoids were infected at least 1 week before transplant, most commonly between day 35 and day 40 of differentiation. Organoids were transferred to a 1.5 ml microcentrifuge Eppendorf tube containing 100 μl neural medium with lentivirus and incubated overnight. The next day, neural organoids were transferred into fresh neural medium in 24-well ultra-low-attachment plates with fresh medium changed daily to wash out any ambient virus particles. All lentiviruses were generated by VectorBuilder with previously used vector maps and published sequences: pLV-hSYN1-GCaMP8s, pLV-hSYN1-oScarlet and pLV-hSYN1-eYFP55,56. For rabies tracing experiments, day 47 hCOs were co-infected with AAV-8-CAG-FLExOFF-RabiesG (AAV-G) from the Stanford viral vector core and G-Deleted Rabies-eGFP (RVΔG-GFP) from Salk Institute. Each organoid was incubated overnight with 0.5 μl of 1:10 diluted RVΔG-eGFP virus and 0.5 μl of AAV-G into 200 μl of the neural medium described above. The next day, 800 μl of the neural media was added to each organoid and, the day after that, the organoids were washed with fresh neural medium three times and transferred into low attachment dishes (6 or 24 well). All cell lines tested negative for mycoplasma contamination, and genomic integrity was assessed using the single-nucleotide polymorphism microarray GSAMD-24v2--0.
Organoid transplantation
All procedures were performed in accordance with NIH guidelines and with previous approval of the Stanford University Administrative Panel on Laboratory Animal Care (APLAC) and in accordance with a previously published protocol13, but optimized for the apallial mouse neuroanatomy. In brief, organoids at 30–60 days in vitro were transplanted into 5–17-day old apallial pups (median, 10; interquartile range, 9–13). Importantly, this is before the critical period of activity-dependent establishment of neural connectivity in the mouse57. Mice were anaesthetized with isoflurane (5% induction, 2–3% maintenance) and received Ethiqa-XR (0.65 mg per kg) or 10 mg per kg carprofen + 0.25–0.5% bupivacaine local infiltration below the incision site before placement into a stereotaxic frame with a neonatal insert (RWD) and heating pad. Switching from Ethiqa-XR to bupivacaine + carprofen helped to reduce the rate of infanticide. While maintaining an intact dura, a craniotomy was performed on each hemisphere at AP, +1 mm; ML ± 1 mm. For each hemisphere, two hCOs were loaded into the Hamilton syringe and the contents of the syringe were deposited at a rate of 10 µl min−1 at a depth of 1.5 mm below the surface of the dura. This yielded four transplanted hCOs per mouse. As the surgeries occurred at such a young age, there was not enough time to reliably obtain genotyping results before the surgery. Therefore, the presence or absence of the neocortex was confirmed through visual inspection after an initial incision.
MRI monitoring of graft development
MRI scans were performed on the Bruker BioSpec 70/40 small-animal scanner (7.0 Tesla, 300 MHz; Bruker) equipped with a BGA-12S gradient insert (660 mT m−1, 4,570 T m−1 s−1) and interfaced to ParaVision 360 V 3.6. A 4-element receive-only mouse CryoProbe and 86 mm volume coil were used for MR imaging. Animals were anaesthetized with 3% isoflurane mixed in O 2 and maintained with 1–1.5% isoflurane during the imaging procedure. Core body temperature was maintained at 37 °C using a warm water circulator pad and a temperature controller (Thermo Fisher Scientific). Mouse respiratory rates were monitored through a pneumatic pad placed underneath the animal (SA instruments).
Two-dimensional T2W Turbo rapid acquisition with relaxation enhancement (RARE) sequence was used to obtain transverse structural MRI for volume measurements (TR/TE = 4,500/41 ms, FOV = 18 × 18 mm2, matrix = 258 × 258 yielding 70 × 70 μm2 in-plane resolution, slice thickness = 0.5 mm, RARE Factor = 8, NEX = 2, scan time = 4 min 3 s). For some animals, 3D RAREvfl sequence was used (TR/TE = 1,800/90.26 ms, FOV = 15 × 15 × 8 mm3, matrix = 150 × 150 × 80 yielding 0.100 mm isotropic resolution, RARE Factor = 43, NEX = 1.6, scan time = 10 min 33 s with compressed sensing) and data are reconstructed using the Smart Noise Reduction option.
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