Embryonic fibroblast isolation
Homozygous TauEGFP knock-in mice 21
were purchased from the Jackson Laboratories and bred with C57Bl6 mice (Taconic) to generate TauEGFP heterozygous embryos. Balb/c mice were purchased from Taconic. Rosa26-rtTA mice were obtained from Rudolf Jaenisch28
. MEFs were isolated from E14.5 embryos under a dissection microscope (Leica). The head, vertebral column (containing the spinal cord), dorsal root ganglia, and all internal organs were removed and discarded to ensure the removal of all cells with neurogenic potential from the cultures. The remaining tissue was manually dissociated and incubated in 0.25% Trypsin (Sigma) for 10–15 minutes to create a single cell suspension. The cells from each embryo were plated onto a 15cm tissue culture dish in MEF media (Dulbecco’s Modified Eagle Medium (Invitrogen) containing 10% Fetal Bovine Serum (FBS) (Hyclone), beta-mercaptoethanol (Sigma-Aldrich), non-essential amino acids, sodium pyruvate, and penicillin/streptomycin (all from Invitrogen). Cells were grown at 37oC for 4–7 days until confluent, and then split once before being frozen. After thawing, cells were cultured on 15cm plates and allowed to become confluent before being split onto plates for infections using 0.25% Trypsin. Postnatal tail tip fibroblasts were prepared by removing the bottom third of tail from 3-day-old pups using surgical scissors. Cells were rinsed in ethanol, washed with HBSS (Sigma), and then dissociated using scissors and 0.25% Trypsin. Tail tip fibroblasts were cultured in MEF media until confluent and passaged once before being pooled together and frozen down for further use.
Cell culture, molecular cloning, and viral infections
We had three criteria for identifying candidates with neuron-inducing activity: (i) we reasoned that cell-fate inducing factors should be enriched in the gene category of transcriptional regulators. (ii) We included factors previously involved in reprogramming to pluripotency (Klf4, c-Myc, and Sox2). (iii) We searched for genes specifically expressed in neural tissues. Those) were selected based on published expression arrays of MEFs, ES cells and neural progenitor cells retrieved from the Gene Expression Omnibus database (GSE8024, http://www.ncbi.nlm.nih.gov/gds
) and the EST Profile function of NCBI’s Unigene database (http://www.ncbi.nlm.nih.gov/unigene
). cDNAs for the factors included in the nineteen factor pool were cloned into lentiviral constructs under the control of the tetracycline operator 35
. Replication-incompetent, VSVg-coated lentiviral particles were packaged in 293T cells as described35
. Passage three TauEGFP and Balb/c MEFs were infected in MEF media containing polybrene (8 μg/mL). After 16–20 hours in media containing lentivirus, the cells were switched into fresh MEF media containing doxycycline (2 μg/mL) to activate expression of the transduced genes. After 48 hours in MEF media with doxycycline (Sigma), the media was replaced with N3 media 22
containing doxycycline. The media was changed every 2–3 days for the duration of the culture period. For BrdU experiments, 10μM BrdU was added to the culture media and was maintained throughout media changes until the cells were fixed.
Immunofluorescence, RT-PCR, and flow cytometry
Neuronal cells were defined as cells, which stained positive for Tuj1and had a process at least 3 times longer than the cell body. For immunofluorescence staining, cells were washed with PBS and then fixed with 4% paraformaldehyde for 10 minutes at room temperature (RT). Cells were then incubated in 0.2% Triton X-100 (Sigma) in PBS for 5 minutes at RT. After washing twice with PBS, cells were blocked in a solution of PBS containing 4% BSA, 1% FBS for 30 minutes at RT. Primary and secondary antibodies were diluted in a solution of PBS containing 4% BSA and 1% FBS. Fields of cells for staining were outlined with a PAP pen (DAKO). Primary and secondary antibodies were typically applied for 1 hour and 30 minutes, respectively. Cells were washed three times with PBS between primary and secondary staining. For anti-BrdU staining, cells were treated with 2N HCl in PBS for 10 minutes and washed twice with PBS before permeablization with TritonX-100 (Sigma). The following antibodies were used for our analysis: goat anti-ChAT (Millipore, 1:100), rabbit anti-GABA (Sigma, 1:4000), rabbit-GFAP (DAKO, 1:4000), mouse anti-MAP2 (Sigma, 1:500), mouse anti-NeuN (Millipore, 1:100), mouse anti-Peripherin (Sigma, 1:100), mouse anti-Sox2 (R&D Systems, 1:50), rabbit anti-Serotonin (Biogenesis, 1:1000), rabbit anti-Tuj1 (Covance, 1:1000), mouse anti-Tuj1 (Covance, 1:1000), goat anti-Brn2 (Santa Cruz Biotechnology, 1:100), mouse anti-BrdU (Becton-Dickinson, 1:3.5), mouse anti-Calretinin (DAKO, 1:100), sheep anti-Tyrosine Hydroxylase (Pel-Freez, 1:1000), E028 rabbit anti-synapsin (gift from Thomas Südhof, 1:500), guinea pig anti-vGLUT1 (Millipore, 1:2000), mouse anti-GAD6 (Developmental Studies Hybridoma Bank (DSHB), 1:500), mouse anti-Pax3 (DSHB, 1:250), mouse anti-Pax6 (DSHB, 1:50), mouse anti-Pax7 (DSHB, 1:250), mouse anti-Nkx2.2 (DSHB, 1:100), mouse anti-Olig1 (NeuroMab, 1:100). Fitc-, and Cy3-conjugated secondary antibodies were obtained from Jackson Immunoresearch. Alexa-488, Alexa-546 and Alexa-633-conjugated secondary antibodies were obtained from Invitrogen. TauEGFP expressing cells were analyzed and sorted on a FACS Aria II (Becton Dickinson). Flow cytometry data was analyzed using Flowjo (Tree Star). After sorting, cells were plated on cortical cultures or glial cultures derived from neonatal brains. Cells were kept in 50% N3 media and 50% growth media (see media composition below) and 2 μg/ml doxycycline for one week before being switched to growth media without doxycycline until electrophysiological analysis was completed. For RT-PCR analysis, RNA was isolated using Trizol (Invitrogen) following the manufacturer’s instructions, treated with DNAse (NEB) and 1.5 μg was reverse-transcribed with Superscript II (Invitrogen). PCR was performed using the following primers Sox1 (F- TCGAGCCCTTCTCACTTGTT, R-TTGATGCATTTTGGGGGTAT), Sox10 (F- GAACTGGGCAAGGTCAAGAA, R-CGCTTGTCACTTTCGTTCAG), β-Actin (F-CGTGGGCCGCCCTAGGCACCA, R-CTTAGGGTTCAGGGGGGC). PCR products were analyzed on a 1% gel.
The following method was used to calculate the efficiency of neuronal induction. The total number of Tuj1+ cells with a neuronal morphology, defined as cells having a circular, three dimensional appearance that extend a thin process at least three times longer than their cell body, were quantified twelve days after infection. This estimate was based on the average number of iN cells present in 30 randomly selected 20× visual fields. The area of a 20× visual field was then measured, and we used this estimated density of iN cells to determine the total number of neurons present in the entire dish. We then divided this number by the number of cells plated before infection to get a percentage of the starting population of cells that adopted neuron-like characteristics.
Primary cortical neurons were isolated from newborn wildtype mice as described 36
with modifications. Briefly, cortices were dissociated by papain (10 U/ml, with 1 μM Ca2+
, and 0.5 μM EGTA) digestions and plated on Matrigel coated circle glass coverslips (Ø 11 mm). The neurons were cultured in vitro in growth media consisting of: MEM (Invitrogen) supplemented with B27 (Invitrogen), glucose, transferrin, FBS and Ara-C (Sigma).
Glial cell isolation
Forebrains were dissected from postnatal day five wild-type mice and were manually dissociated into ~.5 mm2 pieces in a total of 2 mL of HBSS. 500 μL of 2.5% Trypsin and 1% DNase were added and dissociated tissue was incubated at 37° for 15 minutes. Solution was mixed every 5 minutes. The supernatant was then transferred into 1.5 mL of FBS. 4 mL of HBSS, 500 μL 2.5% Trypsin, and 500 μL DNase were again added to the remaining dissociated tissue and incubated at 37° for 15 minutes, mixing every 5 minutes. The supernatant was again removed and added to the FBS-containing solution. Using a pipette, the remaining tissue was further dissociated and passed through a 70 μM nylon mesh filter (BD Biosciences) into the FBS-containing solution. The cell mixture was then spun at 1000 rpm for 5 minutes and resuspended in MEF media. Glial cells were passaged three times before culturing with MEF or TTF-derived iN cells. Contaminating neurons in p3 glial cell cultures could not be detected when stained for either Tuj1 or MAP2.
Recordings were performed from MEF- and tail cell-derived iN cells at 8, 12 and 20 days after viral infection, or 7–13 days after co-culturing with cortical neurons. Spontaneous or evoked synaptic responses were recorded in the whole-cell voltage-clamp mode. Evoked synaptic responses were triggered by 1 ms current injection through a local extracellular electrode (FHC concentric bipolar electrode, Catalogue No. CBAEC75) with a Model 2100 Isolated Pulse Stimulator (A-M Systems, Inc.), and recorded in whole-cell mode using a Multiclamp 700B amplifier (Molecular Devices, Inc.) 37
. Data were digitized at 10 kHz with a 2 kHz low-pass filter. The whole-cell pipette solution for synaptic current recordings contained (in mM): CsCl 135, HEPES 10, EGTA 1, Mg-ATP 4, Na4
GTP 0.4, and QX-314 10, pH 7.4. The bath solution contained (in mM): NaCl 140, KCl 5, CaCl2
0.8, HEPES 10, and glucose 10, pH 7.4. IPSCs were pharmacologically isolated by addition of 50 μM D-AP5 and 20 μM CNQX to the bath solution. EPSCs were pharmacologically isolated by addition of 30 μM picrotoxin and 50 μM D-APV. Data were analyzed using Clampfit 10.02 (Axon Instruments, Inc). Action potentials (APs) were recorded with current-clamp whole-cell configuration. The pipette solution for current clamp experiments contained (in mM) 123 K-gluconate, 10 KCl, 1 MgCl2
, 10 HEPES, 1 EGTA, 0.1 CaCl2
, 1 K2
ATP, 0.2 Na4
GTP, and 4 glucose, pH adjusted to 7.2 with KOH. Membrane potentials were kept around −65 to −70 mV, and step currents were injected to elicit action potential. Whole-cell currents including sodium currents, potassium currents were recorded at a holding potential of −70 mV, voltage steps ranging from −80 mV to +90 mV were delivered at 10 mV increments.