Selective conversion of fibroblasts into peripheral sensory neurons.

Embryonic fibroblasts isolation and derivation Wild-type CD1 mice were bred at The Scripps Research Institute animal facility. MEFs were isolated from E14.5 embryos under a dissection microscope. The head, internal organs and spinal column containing the dorsal root ganglion was removed and discarded to eliminate cells with neurogenic potential. The remaining tissue was manually disassociated in 0.25% trypsin (vol/vol, Gibco) for 10 min at 37 °C subsequently the digestion solution was diluted and removed via centrifugation and subsequently seeded onto tissue culture plates. MEFs were grown to confluence and passaged at least twice before use. For HEF differentiation, human iPSCs colonies were harvested using 1 mg ml −1 collagenase type IV and differentiated by embryoid bodies (EBs) formation. The EBs were cultured for 7 d in non-adherent suspension culture dishes (Corning), 2 d in 20% KSR medium and the following 5 d in 10% FBS DMEM (vol/vol). On day 8, the EBs were plated onto adherent tissue culture dishes and passaged according to primary fibroblast protocols using trypsin for three passages before the start of experiments. Molecular cloning, cell culture and lentiviral infection The cDNAs for human BRN3A (97% homologous to mouse Brn3a peptide) and mouse Ngn1 and Ngn2 were cloned into lentiviral constructs under the control of tetracycline operator (TetO) using the following primers: BRN3A forward and reverse, respectively, 5′-ATGATGTCCATGAACAGCAAGCAG and 5′-TCAGTAAGTGGCAGAGAATTTC. Ngn1 forward and reverse, respectively, 5′-ATGCCTGCCCCTTTGGAGACC and 5′-TTCAGCGAGGGTGCAGCAACC. Ngn2 forward and reverse, respectively, 5′-ATGTTCGTCAAATCTGAGACTCT GG and 5′-AAACCAGAGCTGGTCTCCACC. Replication-incompetent VSVg-coated lentiviral particles were packaged in 293T cells as previously described 33 . Passage three CD1 MEFs were infected with lentivirus in MEF media (DMEM + 10% FBS and penicillin/streptomycin). After 12–16 h of infection media-containing virus was replaced with fresh MEF media. Transcription factors were induced 48 h post infection media by switching to MEF media supplemented with 5 μg ml −1 dox (Sigma). On day 4, media was changed with N3 media with 5 μg ml −1 dox (Sigma) 26 . 7 d post induction, dox was withdrawn unless otherwise stated. 10 d post-induction, media was replaced with neural maintenance media, which consisted of a 1:1 mix of DMEM/F12 (Invitrogen) and Neurobasal supplemented with B27, and NGF, BDNF and GDNF, all at 10 ng ml −1 . Efficiency of conversion was measured by the number of Map2-positive cells divided by to the initial number of cells plated. In experiments with very low conversion efficiencies, we observed that reprogrammed cells exhibited neuronal morphology and marker expression that differed from the results of typical experiments, perhaps as a result of infrequent coexpression of the reprogramming factors or to other conditions related to cell culture. Thus, only experiments with conversion efficiency greater than 1% of starting population were analyzed. Immunohistochemistry, and RT-PCR For immunofluorescence staining, cells were fixed 4% paraformaldehyde (vol/vol) for 10 min at 20–24 °C. Cells were then washed three times with phosphate-buffered saline (PBS) and subsequently permeabilized with 0.1% Triton X-100 (vol/vol, Sigma) in PBS. After washing and permeabilization, cells were blocked in 5% horse serum (vol/vol) for 30 min at 20–24 °C. Primary staining was performed overnight at 4 °C in block. Secondary antibodies were diluted in blocking solution and stained at 20–24 °C for 1 h. EdU staining was performed using Click-it EdU kit ( C10337 , Invitrogen) following manufacturer’s instructions. The following antibodies and dilutions were used: Ms-βIII-Tubulin (Tuj1) (1:1,000, Covance MMS-435P), Rb-βIII-Tubulin (Tuj1) (1:1,000, Covance MRB-435P), Ms-Map2 (1:500, BD 556320), Ms-VGlut1 (1:100, Millipore MAB5502), Rb-Vglut2 (1:50, abcam ab72310), Gp-VGlut3 (1:1,000, Millipore AB5421), Ms-Brn3a (1:200, Millipore MAB1585), Gt-human Ret (1:100, R&D AF1485), Gt-mouse Ret (1:100, R&D AF482), Ms-Islet1 (1:200, DSHB 40.2D6), Gt-mouse TrkB (1:200, R&D BAF1494), Gt-TrkA (1:200, R&D AF175), Sh-TrkC (1:200, Abcam ab72120), Ms-NF200 (1:200, Millipore MAB5266), Rb-Peripherin (1:200, Millipore AB1530), Rb-Gaba (1:500, Sigma A2052), Rb-Ngn1 (1:500, Abcam 66498), Rb-Ngn2 (1:500, Millipore 5682), Rb-P75 (Abcam 8874), Rb-CGRP (1:500, Neuromics RA24112), Ms-VAMP (1:200, Synaptic systems 104 211), Rb-Synapsin (1:1,000, E028). Secondaries: Life Technologies: antibody to goat (1:1,000, A21447D-G647), antibody to mouse (1:1,000, A10036-DM546), antibody to mouse (1:1,000, A21202-DM488), antibody to rabbit (1:1,000, A10040-DR546), antibody to sheep (1:1,000, A11015-DSh488), antibody to goat (1:1,000, A11056-DGt546), antibody to rabbit (1:1,000, A21206-DRb488), antibody to rat (1:1,000, A21208-DRt488) and antibody to sheep (1:1,000, A21098-DSh546). For RT-PCR analysis, total RNA was isolated at the time points indicated using Trizol (Invitrogen) following manufacturer’s instructions, treated with DNaseI (Ambion) and 1.0 μg was reverse transcribed with iScript (BioRad). PCRs were performed using TaqMan Gene Expression Assays (Applied Biosystems) or SYBR green. For quantitative RT-PCR from single cells, single cells were grown on glass coverslips from which they were isolated 3 weeks after induction using a using a patch pipet and micromanipulator. Cells were placed in 4 μl of lysis/RT buffer consisting of Superscript III RT buffer (Invitrogen) supplemented with 0.5% (vol/vol) NP-40, 1 mM DTT, and SuperRnase inhibitor (Ambion), and Prime RNase inhibitor (5 Prime). Cells were spun down in a microcentrifuge and flash frozen at −80 °C until further processing. Reverse transcription was performed using SuperScript III (Invitrogen) with 130 nM of each gene-specific 3′ primers. Reverse transcription products were than subjected to 15 cycles of target specific pre-amplification using 15 nM of outside nested primers designed to produce amplicons of 300–400 bp. Quantitative real-time PCR was subsequently performed using SYBR select (Applied Biosystems, 4472918) with internal primers designed to generate amplicons approximately 100 base pairs. To ensure specificity, template titrations were performed and only primers that demonstrated linear amplifications were used. Melt curves were also obtained for single cells and controls to ensure specificity of products.

Statistics

For samples assumed to have a normal distribution, we applied one-way ANOVA with Newman-Keuls multiple comparison test. In Figure 3a , populations were determined not to have a normal distribution using the D’Agostino & Pearson omnibus test. The comparisons between populations without normal distribution used the non-parametric Kruskal-Wallis test. For Figure 5d , we used an unpaired Student’s t test; data distribution was assumed to be normal, but was not formally tested. No statistical methods were used to pre-determine sample sizes, but our sample sizes are similar to those reported in previous publications 21 , 22 , 25 , 27 .

Calcium imaging

Calcium imaging was performed on mouse and human iSNs 2–3 weeks post-induction using Map2::GCAMP5.G lentiviral reporter 46 . Imaging was performed in Tyrode’s solution (145 mM NaCl, 2.5 mM KCl, 10 mM Hepes, NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Glucose, and 0.4 mM ascorbic acid) at a constant flow rate of 250 ml h −1 . To monitor calcium response, capsaicin, menthol and mustard oil were added sequentially in randomized orders to the flow chamber at a 10× concentration to deliver a final concentration of 10 μM capsaicin, 100 μM menthol and 100 μM mustard oil. Each tracing experiment was bracketed by an initial and final pulse of 25 mM KCl to confirm neural identity and sustained functional ability. Only cells with neural identity and sustained functional ability were analyzed. Calcium responses were determined by calculating the change in fluorescence over the initial fluorescence ( F − F 0 )/ F 0 , where F = the fluorescence at a given time point and F 0 = the mean basal, unstimulated fluorescence of each cell. A typical non-response area was selected for fluorescence bleed normalization and background subtraction. The threshold for a positive calcium response to the addition of a ligand was determined as 1 ( F − F 0 )/ F 0 greater than 0.03 in a 7.4-s window. The intensity threshold for a positive GCAMP response at ( F − F 0 )/ F 0 > 0.03 was determined as 5 s.d. above the mean of s.d. from ( F − F 0 )/ F 0 recordings in BN1 and BN2 neurons during a period of no stimulation ( n = 20). The 7.4-s response window was determined as one s.d. above the mean response time from KCl addition to peak GCAMP intensity in BN1 and BN2 neurons ( n = 30).

Electrophysiology

Fibroblasts were plated, transduced and cultured on laminin coated thermanoxplastic coverslips (13 mm) as described in cell culture methods. Coverslips were placed in the recording chamber mounted on a Nikon Eclipse FN microscope. Spontaneous activity and evoked responses were recorded at 33 °C under whole-cell patch clamp. Signals were amplified using a MultiClamp700B (Molecular Devices) and acquired at 20 kHz using the data acquisition software DASYLab v.11 (National Instruments). Patch pipettes were pulled from standard wall glass of 1.5-mm OD (Warner Instruments) and had input resistances of 6–8 MOhm. The composition of the bath solution (artificial cerebrospinal fluid) was 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 25 mM glucose. For recording action potentials and voltage-gated currents, patch electrodes were filled with the following solution 120 mM potassium-glutonate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, 2 mM MgATP, 0.3 mM Na 3 GTP; pH 7.3. To record voltage responses of the iPSCs, we used incrementing levels of current steps of 350-ms duration. Initial current step level was −50 to −200 pA depending on the observed input resistance of the cell and the steps were incremented by +5 pA. Analysis of the evoked responses was performed in software developed by A. Szücs (IVAnalyzer). Several physiological parameters including the resting membrane potential, rheobase, input resistance at rest, spike amplitude and half-width among others were determined for each cell. To monitor voltage-gated membrane currents, we applied step commands of 50-ms duration and ranging from −60 to +10 mV. In experiments with TTX application, we monitored Na + currents at −20 mV voltage level using 8-s interstimulus intervals. Outward K + currents at −20 mV command level were generally minor relative to the inward Na + component. Leakage correction was performed digitally by using a negative step (−90 mV) preceding each stimulus command. In experiments characterizing TTX resistivity of Na + currents, we applied TTX at 100–300 nM concentrations in bath and recorded control levels, TTX effect and washout in the same file. A Supplementary methods checklist is available.