Derivation of ES-derived neural progenitors
The two cell lines used for generating ESNPs were the Sox1-GFP/ubiquitin RFP ES cell line (Germain et al, in press
) and the parental Sox1-GFP ES line (Ying and Smith, 2003
). The Sox1-GFP/ubiquitin RFP ESNPs were use for transplants, GFP expression is transient but the constitutive RFP expression in this cell line allowed us to identify the ES-derived cells after transplantation. Unless otherwise specified, all media components were purchased from Sigma. The cells were cultured on 0.1% gelatin-coated, culture dishes (Corning Incorporated) in the ES media composed of GMEM containing 10% fetal bovine serum (Atlanta Biologicals), 1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 100 u/ml penicillin/streptomycin (Invitrogen), 0.1 mM β–mercaptoethanol, and LIF (leukemia inhibitory factor derived from CHO-LIF cells). The day of ES cell plating was designated day 0. Pluripotent ES cells were passaged every 2 days.
To generate neural stem cells, ES cells were trypsinized, plated as a monolayer on 0.1% gelatin-coated culture dishes (Corning Incorporated) at a density of 0.5–1.5 × 104 cells/cm2, then differentiated in N2B27 media containing neural basal media (Invitrogen) and DMEM/F12 (1:1 ratio), supplemented with 1% B27 (Invitrogen), 100 u/ml penicillin/streptomycin (Invitrogen), 2 mM L-glutamine, 1% Insulin-Transferrin-Selenite (Invitrogen), 25 μg/ml BSA, 6 ng/ml progesterone, 16 μg/ml putrescine and 0.1 mM β–mercaptoethanol. The neural stem cells were frozen in liquid nitrogen at the stage when ~ 80% were GFP+, ~7 days after plating (day 9).
Neural stem cell differentiation into ESNPs
Neural stem cells were thawed and plated onto laminin-coated, 2-well glass chamber slides (Lab-Tek II) at a density of 6 × 105 cells/ml and grown in N2B27 media supplemented with 2.5 nM Hh agonist (Cur199567; Curis Inc., Cambridge, MA) for 1 day, to aid survival and promote ventral forebrain progenitor fates. The Hh agonist-containing media was replaced with N2B27 for 1 day (on day 11) and the cells were then replated onto laminin-coated 2-well and 8-well chamber slides at a density of 3 × 105 cells/ml in N2B27 media containing Hh agonist for 1 additional day. The cells were grown in N2B27 for two additional days before harvesting on day 14 for transplantation.
RT-PCR and RT-qPCR analyses
To analyze the expression of transcription factors in the cultures, ESNPs or ES cells were dissociated in ultraspec-RNA (Biotecx) and analyzed by Reverse-Transcriptase PCR (RT-PCR) or real time quantitative PCR (RT-qPCR). Total mRNA was extracted from the cells after plating on days 2, 9, 11, and 14. For comparison, fetal mouse MGE mRNA extracts were obtained from 6 CD-1 embryos at embryonic day 13.5 (E13.5) (Charles River). Cells were dissociated in ultraspec-RNA and incubated in Turbo DNA-free (Applied Biosystems). The mRNA concentration was measured by spectrophotometry (Nanodrop Spectrophotometer, Thermo Scientific). Reverse transcription was performed on 10-μg mRNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The primers (Integrated DNA Technologies) are shown in .
Primers used for PCR analyses.
RT-qPCR analyses were carried out with TaqMan primers (Applied Biosystems, Inc.). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH
; Mm99999915_g1) was used as a loading control. The following genes were tested: microtubule-associated protein 2 (MAP2
; 91 bp, Tm=60 °C, Mm00485230_m1), glutamic acid decarboxylase 2 (GAD2
; 99 bp, Tm=60 °C) (Chatzi et al., 2009
), distal-less homeobox 2 (Dlx2
; Mm00438427_m1), and paired box gene 6 (Pax6;
Mm00443081_m1). RT-qPCR results were averaged from triplicate samples processed in parallel and the results were replicated in three separate sets of experiments. Data analyses were performed with SDS v1.2x System Software (Applied Biosystems).
Immunohistochemical analyses of ES-derived neural progenitors in vitro
Cultures of ESNPs were maintained until day 14 in 8-well chamber slides and fixed at RT in 4% paraformaldehyde (PFA) in 0.1 M phosphate (PO4), then rinsed in phosphate buffered saline (PBS, pH 7.4) and stored in anti-freeze medium consisting of 30% ethylene glycol and 30% glycerol in 0.1 M PO4 until immunostaining was carried out. Tissue sections were permeabilized with 0.3% Triton X-100 and blocked in 0.1% Tween-20, 2% BSA, and 5% normal goat serum in PBS, prior to staining in one or more of the following antibodies (diluted 1:1000 unless otherwise noted): mouse anti-nestin (Chemicon), rabbit anti-phospho-histone H3 (Upstate), mouse anti-MAP 2 (Sigma), mouse anti-β-3 tubulin (1:2000, Babco), mouse anti-Mash1 (1:500, BD), rabbit anti-Tbr 2 (Upstate), rabbit anti-CR (Millipore), and rabbit anti-CB (Swant). The staining was detected with species appropriate secondary antibodies including: goat anti-mouse IgG-Alexa 488, goat anti-mouse IgG-Alexa 647, or goat anti-rabbit IgG-Alexa 568 (1:1000 dilution, Molecular Probes). Controls consisted of experiments in which the primary antibodies were omitted. Due to negligible expression of Sox1-GFP in ES cell derivatives after fixation, immunodetection of nestin, MAP 2 and β-3 tubulin were performed using Alexa 488-conjugated secondary antibodies. Quantification of cellular expression of nestin, phospho-histone H3 (H3), MAP 2, β-3 tubulin, or Mash1 was determined by calculating the percentage of cells expressing the specific molecular marker divided by total cell population; quantifications for CB and CR were determined by calculating the percentage of cells expressing the marker divided by the number of MAP2+ cells.
Pilocarpine-induced status epilepticus in mice
The Wesleyan IACUC, in accordance with PHS Policy on Humane Care and Use of Laboratory Animals, approved all procedures involving animals. Six to 8 week old C57Bl/6 adult male mice from Harlan Labs were individually housed and handled daily for two weeks prior to experimentation. To induce seizures, the mice were injected with 0.07 cc of 0. 5-mg/ml-atropine methyl nitrate in sterile saline (i.p. Sigma). Thirty minutes later, the mice were injected with pilocarpine in sterile saline (280 mg/kg, i.p. Sigma). Seizures were scored by a modified Racine scale (Shibley and Smith, 2002
) and status epilepticus (SE) was defined as 3 or more stage 5 seizures. After one hour of SE, the seizures were attenuated with diazepam (10 mg/kg, i.p. Henry Schein). The mice were injected with sterile Ringer’s solution (s.c. Henry Schein), and returned to their home cages. For this study, we induced seizures in 62 mice, 36 of them reached SE (58%), and 21 survived (34%) for up to 4 months. Nineteen mice died during seizure induction (30%). Of the surviving SE mice, 16 received ESNP transplants (). The immunosuppressant cyclosporine A (100 mg/L, Calbiochem) was supplied to the mice in the drinking water beginning two days before transplantation and continued until euthanasia.
Analysis of ESNP transplants.
Stem cell transplantation
Two weeks following induction of SE, adult male mice were anesthetized with isoflurane and injected bilaterally with approximately 100,000 cells in 1 μl of N2B27 media per hippocampus, using a digital stereotaxic apparatus (Kopf) outfitted with a glass syringe and a 22 gauge needle with a 30 degree bevel (Hamilton). The stereotactic coordinates for the dentate gyrus of the dorsal hippocampus were AP: 2.5 mm, ML: ± 2.1 mm, DV: 2.0 mm. Following stereotaxic injections, the needle was left in place for 5 minutes before being withdrawn. The surgical incisions were sealed using 3M Vetbond Tissue Adhesive (Henry Schein) and the mice were monitored during recovery in their original cages.
Perfusion and immunohistochemistry
Eight to 10 weeks after receiving ESNP grafts, one group of mice was euthanized to examine the phenotypes of the transplanted ESNPs. Mice were overdosed with sodium pentobarbital (200 mg/kg i.p. Abbott Labs) and perfused with 4% PFA in 0.1 M PO4, pH 7.4, containing 1μg/ml heparin (Hospira, IL) and 10% sucrose. The brains were postfixed in 4% PFA overnight at 4 °C, equilibrated in sucrose, embedded in tissue freezing media (Triangle Biomedical Sciences) and stored at −80 °C. Cryostat sections were cut at 12 μm intervals in the coronal or horizontal planes, thaw-mounted onto Superfrost Plus slides, and stored at −80 °C.
For immunohistochemical analyses, cryostat sections were permeabilized in 0.3% Triton-X-100 for 20 minutes and incubated for 1 hour in blocking buffer containing 0.1% Tween-20, 2% BSA, and 5% normal goat serum. The sections were then incubated at 4 °C for 12–18 hours in primary antibodies. The following primary antibodies were used (1:1000 dilution, unless otherwise noted): rabbit anti-RFP (Rockland), rabbit anti-SOM (Bachem), rabbit anti-PV (Sigma), mouse anti-NeuN (1:500, Chemicon), rabbit anti-CB (Swant), mouse anti-CR (Swant), and rat anti-ctip2 (1:500, Abcam). For PV and SOM staining, heat-induced epitope antigen retrieval in citric acid buffer (pH 6) was performed before permeabilization. Goat anti-rabbit IgG-Alexa 568 was used to detect RFP in combination with goat anti-mouse IgG-Alexa 488, goat anti-rat IgG-Alexa 488 or goat anti-mouse IgG Alexa-647. For PV, SOM, and CB/ctip2 staining, we omitted the rabbit anti-RFP antibody, as the unamplified fluorescent signal from the cells was sufficient for microscopic detection. Nuclear staining was performed with Hoechst 33342 (1:10,000, Molecular Probes) or Sytox green (1:10,000, Molecular Probes). To quantify the proportions of transplanted cells expressing different molecular markers, three brain sections containing the transplanted cells were counted from each mouse. The sections were ~100 μm apart and counts were made in a minimum of 4 mice. Data were expressed as the percentage of cells expressing the antigen/total RFP+ cells. The standard errors of the means were calculated.
Mossy Fiber Sprouting
Dual immunofluorescent staining of mossy fiber sprouting and transplanted ESNPs was carried out in vibratome sections of the hippocampus that were fixed, cryoprotected and sectioned at 12 micron thicknesses and mounted onto slides. Antigen retrieval was performed in citric acid buffer, and endogenous peroxidase activity was quenched with 0.5% hydrogen peroxide in sodium phosphate buffer. The sections were then incubated in blocking reagent (TNB; Perkin Elmer) followed by incubation in primary antibody mixture consisting of rabbit anti-RFP and guinea pig anti-zinc transporter T3 (ZnT3; 1:200, Synaptic Systems) to label mossy fibers. Detection of the guinea pig primary antibody was performed with goat anti-guinea pig IgG-conjugated to HRP (1:1000, Invitrogen). Tyramide signal amplification (TSA) with green fluorescence was carried out for 10 min, according to the manufacturer’s instructions (Perkin Elmer). Sections were then incubated at RT for 30 min in goat anti-rabbit IgG-Alexa 568 conjugate (1:500, Molecular Probes), stained for Nissl substance with Neurotrace (1:200, Invitrogen) and mounted in Prolong Antifade reagent containing DAPI. Confocal images were collected on a Zeiss LSM 510 laser-scanning confocal microscope.
Fluorescent in situ hybridization
Immunochemical staining for GABA significantly underestimates the number of GABAergic neurons in the mouse brain (Houser and Esclapez, 1994
) we performed fluorescent in situ
hybridization (FISH) for GAD1
mRNA to obtain accurate estimates. The mice were perfused transcardially as described above and 12 μm thick cryostat sections were prepared. Antisense digoxigenin (DIG)-labeled riboprobes were generated against the 3′ UTR of GAD1
by in vitro
transcription using DIG-11-UTP labeling mix (Roche) and Sp6 polymerase (New England Biolabs). Plasmids containing templates for the two riboprobes were provided by Dr. Ralph DiLeone (Yale School of Medicine).
FISH was performed by dehydrating sections in alcohols and air-drying them. The sections were then acetylated for 10 minutes, dehydrated a second time, and hybridized overnight at 60 °C with 200 ng of antisense riboprobe in hybridization buffer. The riboprobes were denatured at 80 °C for 5 minutes and briefly cooled on ice prior to hybridization. After hybridization, the sections were washed in 5X saline-sodium citrate (SSC) buffer at 65 °C for 30 minutes, then in 50% formamide in 2XSSC for 50 minutes at 65 °C. Sections were then treated with 2X, 0.2X, 0.1XSSC for 5 minutes each at RT, then blocked in 5% normal mouse serum (Jackson ImuunoResearch) for 30 minutes, incubated in HRP-conjugated mouse anti-DIG antibody (1:400, Jackson ImmunoResearch). Sections were then rinsed with TBST, and incubated in fluorescein-coupled tyramide amplification reagents (Perkin Elmer), according to the manufacturer’s instructions. Sections containing transplants were then processed for RFP expression by immunofluorescent staining, as described above and nuclei were stained with Hoechst 33342. Controls included hybridization with sense probes for GAD1/2.
Hippocampal slice electrophysiology
Whole-cell patch-clamp recordings were made in hippocampal slices 8–12 weeks after transplantation. The slices were obtained from 12 adult C57Bl/6J mice16–22 weeks of age. Additional recordings were made from hippocampal slices obtained from adult GIN mice (GFP-expressing Inhibitory Neurons) (Oliva et al., 2000
). GFP in these mice co-localizes with SOM, but not PV. The mice were decapitated under deep anesthesia induced by injection of ketamine/xylazine (120 mg/kg ketamine plus 10 mg/kg xylazine, i.p. Henry Schein). Brains were rapidly removed, transferred to cold, oxygenated artificial cerebral spinal fluid (ACSF with high sucrose, 27.07 mM NaHCO3
, 1.5 mM NaH2
, 1 mM CaCl2
, 3 mM MgSO4
, 2.5 mM KCl, 222.14 mM sucrose) and 350 μm thick slices containing the hippocampus were cut on a vibratome (Leica VT1000S) in the coronal or horizontal plane at a 12.5° angle, descending towards the posterior (Rafiq et al., 1993
Slices were transferred to a chamber containing pre-warmed ACSF (125 mM NaCl, 1 mM CaCl, 3 mM MgSO4, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, 3 mM myo-Inositol, 2 mM Na-pyruvate, 0.4 mM ascorbic acid) and incubated for 1 hour, then placed in the recording chamber. While recording, the slices were perfused with ACSF containing low divalent ions (ACSF, 1.25 mM NaH2PO4, 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 25 mM glucose, 3 mM myo-inositol, 2 mM sodium pyruvate, 0.4 mM ascorbic acid, 1 mM CaCl2, 3 mM MgSO4), and recorded at a temperature of 34°C.
Patch pipettes were pulled (Sutter Instrument Co. Model P-97) with 6–9 MΩ resistance and filled with a solution containing 130 mM potassium-methylsulfonate, 11 mM biocytin, 10 mM potassium chloride, 10 mM HEPES, 5 mM sodium chloride, 2.5 mM Mg-ATP, 0.3 mM Na-GTP, and 0.5% mM biocytin. Analog signals were digitized at 10 kHz with an ITC-18 (Instrutech) and acquired with IGOR software (Wavemetrics). Passive membrane properties and firing properties of the cells were analyzed off line with IGOR software. We measured the action potential (AP) amplitude, half width, and delay to spike (defined as the time from the onset of stimulation to the initiation of the first AP). Spike frequency adaptation was calculated as the interval between last two APs divided by the interval between the first two APs (McGarry et al., 2010
). Voltage sag ratio was calculated as the peak voltage drop against end voltage change in response to negative current injections of 500 ms durations (Haghdoust et al., 2007
). The AP drop (a measure of spike accommodation) was obtained by subtracting the peak amplitude of the second AP from the first AP peak amplitude (McGarry et al., 2010
). The maximum number of APs was counted in response to 80–120 pA stimulations lasting 500 ms. Firing frequency (Hz) was calculated as the number of spikes evoked during the current injection divided by the duration of the spiking period, where the spiking period was defined as the time from the beginning of the current injection to the end of the last action potential evoked during the current injection. Immediately after the recordings were made, the slices were fixed in 4% PFA in 0.1 M PO4
overnight and equilibrated in 30% sucrose for several hours before transferring to anti-freeze media for long-term storage in a −20 °C freezer. Some of these slices were sectioned into 12 μm cryostat sections for immunohistochemical analyses (see ).
All procedures were performed at RT. Brain slices were thawed and rinsed in PBS, then incubated in 0.02 M KPBS for 15 minutes before overnight incubation in a Texas Red Avidin D solution consisting of 2.5 mg/ml Texas Red (Vector Labs) and 0.3% Triton X-100 in 0.02 M KPBS. The next day, the slices were rinsed and mounted in Prolong Antifade reagent (Invitrogen) or VectaShield (Vector Labs) and biocytin staining was visualized under epifluorescence with a Zeiss LSM 510 confocal scanning laser microscope.
For comparisons of relative gene expression profiles and current-clamp recordings of different neuronal types, we performed ANOVA with Tukey’s post hoc analysis in SPSS software (IBM SPSS statistics 19). For analysis of voltage-clamp recordings, we used Matlab software (Mathworks).