Procedimiento de la Acción de Tutela

All animal work was done in compliance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes, under the project licence of Professor Stephen B. Dunnett (PPL 30/3036; PIL I0D92ED42).

2.3.1 Transplantation of hPSC-derived MGE progenitors into neonatal rats (with Claire Kelly)

2.3.1.1 Cell suspension preparation

Cells cultured to day 20-22 of MGE differentiation (SHH and purmorphamine added day 10-18) were washed with PBS and dissociated with accutase (15 minutes at 37°C). N2B27 medium was added to inactivate the accutase and the cell suspension was counted and then centrifuged at 1150 rpm for 3 minutes. Cells were resuspended in DMEM-F12 to a volume for 2 x 105 _{cells per µl and transported on} ice to the surgery room.

2.3.1.2 Postnatal care of mother and pups

Pregnant female Sprague-Dawley rats (Charles River) gave birth to litters and were left 1 day to ensure they had fed and bonded with their pups. To avoid the mothers rejecting their pups after surgery, the mother was removed from her pups before surgery and placed in a separate cage. Once all pups had been injected, the mother was put under isoflurane anaesthesia for 3 minutes and then placed back in her home cage surrounded by her pups.

2.3.1.3 Transplantation procedure

All surgical procedures were carried out under isoflurane anaesthesia using a neonatal adaptor on a stereotaxic frame. Post-natal day 2 Sprague Dawley pups were injected with 2 x 105 _{cells in a volume} of 1 µl using a syringe. Injection coordinates targeted the right cortex or striatum (0.9 mm anterior and 1.8 mm lateral to bregma, 2.0 mm below the dura; 0.7 mm anterior and 1.9 mm lateral to bregma, 2.9 mm below the dura). Cells were injected over 1 minute and the needle was left in place another 3 minutes before withdrawal. The scalp was glued together using Vetbond (3M) and pups were placed in warm recovery boxes for 10 minutes before being put back in their home cage.

2.3.1.4 Experimental groups and time points

Two rounds of transplantation were carried out with two litters per experiment. The first round included 23 pups, sacrificed at 4, 8, 16 and 24 weeks (n=2, 2, 4 and 15). The second round included 31 pups, of which 26 were given daily intraperitoneal Cyclosporine A injections (10 mg/kg; Sandimmun) from weaning onwards, and were sacrificed at 6, 12 and 20 weeks (n=6, 9 and 7; 4 rats had to be sacrificed early due to illness). The remaining 5 animals were not treated with immune suppression and were all sacrificed at 20 weeks.

2.3.2 Tissue preparation

Animals were sacrificed at by giving a lethal dose of Euthatal (Merial) and were trans-cardially perfused with pre-wash (1.8% di-sodium hydrogen phosphate, 0.9% sodium chloride, in ddH2O, pH 7.3) for 2 minutes and PFA (4% diluted in pre-wash) for 5 minutes. The brains were removed and post- fixed in 4% PFA for another 4 hours before being transferred to 25% sucrose (in pre-wash) for storage at 4°C up to 1 week. Coronal sections were cut to 40 µm thickness in 12 series using a microtome (RM2245, Leica) with a freezing stage (BFS-3MP Controller, PhysiTemp). Sections were stored in anti- freeze solution (0.545% di-sodium hydrogen phosphate, 0.157% sodium dihydrogen phosphate, 40% ddH2O, pH 7.3; add 30% ethylene glycol, 30% glycerol) at -20°C for up to 6 months before staining.

2.3.3 Immunohistochemistry

All steps were performed on a shaker at room temperature. Floating sections were washed 3 times for 10 minutes in TBS (1.2% Trizma base, 0.9% sodium chloride, in ddH2O, pH 7.4) and blocked (Triton- X-100-TBS with 3% donkey serum) for 1 hour. Sections were incubated in primary antibody solution (TXTBS + 1% donkey serum + primary antibodies) overnight. Sections were washed 3 times for 10 minutes in TBS before incubation in secondary antibody solution (TBS + secondary antibodies; 1/200, AlexaFluor 488, 555, 594 and 647) overnight. Sections were washed 3 times for 10 minutes in TBS and then put in TNS (0.6% Trizma base in ddH2O, pH 7.4) for mounting onto gelatin-coated glass slides. Sections were air dried, cover-slipped with VectaShield hardset antifade mounting medium with DAPI (Vector Labs) and stored at 4°C. All primary antibodies are listed in Table 2.1.

2.3.4 Section imaging and analysis

Imaging of sections was carried out as described for cells (Section 2.2.1). Absolute cell counts were performed for HuNu (human nuclei) and NKX2.1 and corrected using the Abercrombie formula:

P=1/F*N*M/(D+M)

P = number of cells per graft; N = total raw cell count; F = frequency of sampled sections (1:12); M = section thickness (0.04 mm); D = Mean cell diameter.

NeuN-positive cells were counted as a percentage of GFP and the mean and SEM calculated between subjects. Calretinin and nestin are cytoplasmic markers making reliable counting of cells in clumps using a widefield fluorescent microscope unfeasible. Only cells separate from the clumps were counted for these markers as a percentage of GFP.

2.3.5 Ex vivo brain slice recording (Michael Laing)

Coronal slices (300 µm) containing the striatum or hippocampus were prepared from 12-week-old rats (12 weeks post-transplantation) in chilled (1-3°C) cutting solution bubbled with carbogen (95% O2/5%

CO2) (in mM: 60 sucrose, 85 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 3 kynurenic acid, and 0.045 indomethacin). Slices were stored for 20 min at 35°C in sucrose-containing solution and then maintained at room temperature in aCSF [in mM: 125 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 25 D-glucose (305 mOsm)] and used within 4-6 hours. For recording, slices were transferred to a submersion chamber continuously perfused with warmed (35°C) aCSF [in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 25 D-glucose (305 mOsm)] at a flow rate of 2-2.5 ml/min. Somatic whole-cell patch-clamp recordings were performed on GFP- positive cells (visually identified by LED fluorescence and infrared DIC videomicroscopy through an Olympus BX51 microscope) using pipettes with resistances of 4–6 MΩ when filled with internal solution containing (in mM): 130 K-gluconate, 20 KCl, 10 HEPES, 0.16 EGTA, 2 Mg-ATP, 2 Na2-ATP, and 0.3 Na2-GTP, pH 7.3 (295 mOsm). Electrophysiological data were acquired at 20 kHz and filtered at 6 kHz using a Multiclamp 700B patch-clamp amplifier and Digidata 1550 analogue to digital converter with pClamp 10 software (Molecular Devices). Evoked voltage responses were measured by injecting 500 ms current steps from 0-300 pA. Mean resting membrane potential was calculated using 1 s segments from 10 sweeps. Data were analysed with Clampfit software (Molecular Devices) then exported to and plotted using Origin software (OriginLab, USA). These experiments were performed by Michael Laing and the data analysed by myself.