hESC culture and neural induction
Differentiation of hESCs into neurons was performed using a modification of previously described methods (Elkabetz and Studer, 2008
; Li et al., 2008
; Cho et al., 2011
). Briefly, undifferentiated H9 hESCs were grown on a monolayer of fibroblasts from human foreskin (Hs27, ATCC). Cell culture medium was composed of DMEM/F12, knockout serum replacement (20%), basic fibroblast growth factor (8 ng/ml), β-mercaptoethanol (0.1 mM), and non-essential amino acids (1 mM). Media changes were performed daily and cells were subcultured on a weekly basis. For neural stem/progenitor cell (NPC) induction, hESCs were incubated for 24 hours in cell culture medium composed of DMEM/F12:Neurobasal (1:1), B27 serum-free supplement (2%), and N2 serum-free supplement (1%). They were then mechanically dissociated until small clusters of cells were obtained. These cell clusters were cultured for up to 72 hours in the same medium. For neural expansion, cells were grown for up to 6 days in cell culture medium composed of DMEM/F12:Neurobasal (1:1), B27 serum-free supplement (1%), N2 serum-free supplement (0.5%), basic fibroblast growth factor (20 ng/ml), and epidermal growth factor (20 ng/ml). Cells were then plated on dishes coated with 10 μg/ml laminin and grown for up to 48 hours until they took a rosette-like appearance. These rosette-neural stem cells (R-NSCs) were retrieved using a needle and transferred to a dish where they were cultured for 3 to 30 days in the same medium.
Immunocytochemistry and immunoblots
Epifluorescence microscopy for immunocytochemical analysis and gel electrophoresis followed by immunoblot analysis were performed as previously described (Li et al., 2008
; Cho et al., 2011
). Primary antibodies included: β-tubulin III (TuJ1; mouse, 1:1000, Covance), microtubule-associated protein 2 (MAP2; mouse, 1:1000, Sigma), synaptophysin (SYPH; rabbit, 1:1000, Dako), tyrosine hydroxylase (TH; mouse, 1:1000, Pel-Freez; rabbit, 1:1000, Chemicon), glutamate transporter 1 protein (vGAT; mouse, 1:1000), and glutamic acid decarboxylase (GAD65/67; rabbit, 1:500, Abcam). Alexa 488, 555 or 647-conjugated goat anti-mouse or anti-rabbit IgGs (1:1000, Invitrogen) were used as secondary antibodies.
Lentiviral infection of R-NSCs and transplantation
Cells at the R-NSC stage were transduced with lentiviral expression vectors. As previously described, to enhance neurogenesis we expressed a lentivirus containing a constitutively active form of the transcription factor myocyte enhancer factor-2 (MEF2C-CA) (Li et al., 2008
; Cho et al., 2011
). Additional lentiviral vectors included pSynapsinI-hChR2-EGFP and pEF1-NpHR-mCherry (Zhang et al., 2006
). Once infected, R-NSCs were dissociated and transferred to plates coated with a mixture of 10 μg/ml poly-L-ornithine and 1 μg/ml laminin. These R-NSCs were grown to become hNPCs as a monolayer in the medium for neural expansion. At this point, hNPCs were enzymatically dissociated into a single cell suspension using Accutase. For transplantation, hNPCs were washed in Ca2+
-free Hanks’ buffered salt solution and concentrated. Cells were placed onto the CA3 region using a glass micropipette attached to a nanoliter injector (Nanoject, Drummond). Each slice received a single injection of a single-cell suspension containing 50,000–100,000 cells. Slices were placed back in a 37 °C/5% CO2
incubator and analyzed for electrophysiological activity every week for at least 4 weeks post injection.
Organotypic hippocampal slices
Organotypic slices were prepared as described elsewhere (Stoppini et al., 1991
; Scheffler et al., 2003
; Opitz et al., 2007
). Briefly, 400 μm-thick transverse hippocampal slices were cut from P6 male and female rats pups (Sprague-Dawley, Harlan), in ice-cold calcium-free MEM. Immediately after sectioning, the slices were transferred on Millicell-CM membrane inserts (Millipore) in wells containing culture medium of the following composition: 50% Basal Medium Eagle, 25% horse serum, 19% Earle’s Balanced Salt Solution, 25 mM Hepes, 32 mM glucose; 2 mM glutamine, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B, pH 7.20. Slices were kept in a humidified incubator at 37 °C in 5% CO2
. After 24 hours, slices were transferred to a maintenance culture medium of similar composition with a lower concentration of horse serum (5%). Media was changed every 3–4 days.
For cultures, whole-cell recordings were performed at room temperature (22 °C) using a patch-clamp amplifier (Axopatch 200B, Molecular Devices, Union City, CA). Cells on coverslips were placed in a 150 μl recording chamber mounted on the stage of a Zeiss Axiovert inverted microscope. Cells were continuously superfused with Hepes-buffered external solution of the following composition (in mM): 137 NaCl, 1 NaHCO3, 0.34 Na2HPO4, 2.5 KCl, 0.44 KH2PO4, 2.5 CaCl2, 5 HEPES, 22.2 glucose, pH adjusted to 7.3 with NaOH. Patch pipettes were pulled from borosilicate glass capillaries (GC150F-10, Warner Instruments) using a Flaming/Brown micropipette puller (P80/PC, Sutter Instruments, Novato, CA). Patch pipettes had open tip resistances of 4–10 MΩ. For organotypic slices, extracellular field potentials were recorded at 35 °C using a multielectrode array (MEA60, Multi Channel Systems, Reutlingen, Germany). The MEA chamber contained 60 electrodes, each 30 μm in diameter at a distance apart of 200 μm. Slices were held down against the electrodes by a ring with a fine mesh and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 D-glucose; pH 7.4, osmolarity 310 mOsm. Photostimulation of optogenetic constructs was controlled via a xenon lamp in a Lambda DG-4 High Speed Filter Changer (Sutter, Novato, CA), equipped with appropriate excitation filters (ChR2: FF01-475/35-25; NpHR: FF01-585/40-25, Semrock, Rochester, NY).