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Selective conversion of fibroblasts into peripheral sensory neurons

Abstract

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

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Figure 1: Transient coexpression of two developmentally relevant transcription factors stably reprograms fibroblasts to attain properties of functionally mature neurons.
Figure 2: Neurons induced with BN1 or BN2 exhibit molecular hallmarks of the peripheral sensory neural lineage.
Figure 3: Reprogramming induces peripheral sensory neural morphology.
Figure 4: iSNs exhibit functional properties of sensory neurons.
Figure 5: iSN reprogramming does not require proliferating or specialized embryonic precursor.
Figure 6: Human iSNs are generated using BN1 and BN2.
Figure 7: Human iSNs display physiological properties of mature sensory neurons.
Figure 8: Human iSNs possess functional properties of sensory neurons.

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Acknowledgements

We would like to thank M. Talantova and D. Zhang for assistance with mouse electrophysiology, K. Spencer for assistance with microscopy, A. Patapoutian, S. Murthy, A. Dubin, S. Ranade and J. Mathur for helpful discussions, and W. Ferguson for technical assistance. This research was supported by the National Institute on Drug Abuse (DA031566 to P.S.), the National Institute on Deafness and other Communication Disorders (DC012592 to K.K.B.), the National Institute of Mental Health (MH102698 to K.K.B.), the California Institute for Regenerative Medicine (RB3-02186 to K.K.B.), the Baxter Family, Norris and Del Webb Foundations (K.K.B.), by Las Patronas and the Dorris Neuroscience Center (K.K.B.), a pre-doctoral fellowship from the California Institute of Regenerative Medicine (J.W.B. and R.K.T.), an NSF Predoctoral Fellowship (R.K.T.), the Andrea Elizabeth Vogt Memorial Award (J.W.B.) and the Scripps Stem Cell Postdoctoral Fellowship (V.L.S.).

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Contributions

K.K.B., J.W.B. and K.T.E. designed and conceived the experiments. K.T.E., J.W.B., V.L.S., R.K.T. and D.W. performed the experiments. A.S. and P.P.S. performed electrophysiology. K.K.B., J.W.B. and K.T.E. wrote and revised the manuscript and all of the authors edited the final drafts.

Corresponding author

Correspondence to Kristin K Baldwin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Reprogramming methods and controls

(a) Cartoon schematic of doxycycline inducible reprogramming vectors. (b) Schematic of reprogramming methods. Mouse embryonic fibroblasts were obtained from e14.5 embryos. Embryos were carefully dissected to remove the spinal cord and internal organs and passaged at least twice prior to infecting with reprogramming cocktails. Doxycycline (5 μg/ml) was applied for 8 days and then removed after which cells were allowed to mature in culture for various time periods, typically 8 days (c) Co-expression of Brn3a with either Ngn1 or Ngn2 is required for induction of Map2/Tuj1 neural cells. (d) Quantification of immunostaining in parental MEF population. (e) Time course of doxycycline induction of reprogramming factors. Dox was removed at the indicated days. The number of Map2/Tuj1 positive cells was quantified on day 14 (f) Ectopic BN1 or BN2 expression consistently reprograms MEFs into Map2/Tuj1 positive cells with neural morphology. Each experiment represents a different viral preparation. Reprogramming efficiency typically ranged between 1 and 10% of the starting population. The variability in efficiency is likely due to variance in viral titer.

Supplementary Figure 2 Summary of electrophysiological recordings in mouse BN1 and BN2 induced iSNs

(a) Pcdh21::CRE x Ai9 expression (red) in the DRG and in BN1 and BN2 induced neurons was used to identify neural cells for patching. Representative current clamp trace from BN1 cell showing voltage sag. (b) Table showing resting membrane potential (RMP), amplitude of sodium currents, the presence (y) or absence (n) of evoked or spontaneous action potentials, and the genotype. * denotes cells patched during a separate electrophysiology collaboration. For these cells voltage-clamp experiments were not performed. (c) Quantification of mean RMPs and sodium current amplitudes (iNa+). Error bars represent SEM.

Supplementary Figure 3 Characterization of neural cells induced by BN1 and BN2.

(a) Marker and antibody validation on mouse DRG sections. (b) BN1 and BN2 neurons are glutamatergic and express vGlut1, 2, and 3 as well the sensory neuropeptide CGRP. (c) Mature BN1 and BN2 cells maintain expression of Brn3a but down-regulate expression of Ngn1 and Ngn2. (d) Mean Ct value from single cell RT-PCR for each biological group. Error bars represent ±SEM. ***p<0.001; **p<0.01(one-way ANOVA) (Gapdh p = 0.0896; NTC p = 0.0743; Fsp1 p < 2E-16; Snail p < 2E-16; Map2 p < 2E-16; Isl1 p = 8.07E-5) For p values < 2E-16, it was not possible to compute exact values. (e) Left column graphs are mean Ct value of Trk receptors from single cell RT-PCR for each biological group (TrkA p = 0.3571; TrkB p = 0.0139; TrkC p = 0.1088). Error bars represent ±SEM. ***p<0.001; **p<0.01(one-way ANOVA with Newman-Keuls multiple comparison test) right column graphs are 1/Ct plotted for each cell by biological group. (f) Pair-wise immunostaining for Trk receptors labels approximately 60% of neural cells.

Supplementary Figure 4 Expression of Trk receptors and Soma Size distribution in iSNs

(a) Immunostaining for TrkA labeled neural cells with small soma. (b) Immunostaining for TrkB labeled neural cells with larger soma. (c) TrkA, TrkB, and TrkC neurons exhibit distinct somatic area distributions. Scatter plot of soma areas by Trk receptor immunoreactivity. (d) Table comparing soma areas between TrkA, TrkB and TrkC populations generated with BN1 or BN2

Supplementary Figure 5 iSNs exhibit pseudounipolar morphology characteristic of peripheral sensory neurons

Representative images of neurite morphology for neurons induced with Brn2, Ascl1 (also called Mash1) and Zic1 (BAZ) and BN1 and BN2. Black is Tuj1 staining. Scale bars of 100 μm apply to all panels

Supplementary Figure 6 Neurogenins play a specific role in specifying induced sensory neurons

Ngn1 and Ngn2 are critical for Trk receptor expression in the iSN reprogramming cocktail. (a) Reprogramming efficiencies between pair-wise combination of Brn3a with Ngn1, Ngn2 or Ascl1 did not vary significantly (p = 0.2147). (b) Ngn1 or Ngn2 are required for Trk receptor expression. Percentage of total Map2 and Tuj1 positive neurons coexpressing each Trk receptor in induced neurons generated from Brn3a with either Ngn1, Ngn2, or Ascl1. **p<0.001 (BN1 vs. BN2 p = 0.8481, BN1 vs. Brn3a/Ascl1 p = 0.0002, BN2 vs Brn3a/Ascl1 p = 0.0003). (c) Graph quantifies percentage of neurons with multipolar, bipolar, or unipolar neurite structure in induced neurons generated from Brn3a paired with Ngn1, Ngn2, or Ascl1. (d) Representative images for neurons induced by each pair-wise combination, each were taken with the same parameters and the same magnification. Black is Tuj1 staining

Supplementary Figure 7 iSNs exhibit physiological properties of peripheral sensory neurons

(a) Quantitative RT-PCR of MEFs, neurons induced with BAZ and iSNs generated with BN1 or BN2. BAZ, BN1 and BN2 samples expressed similar levels of Map2. In contrast, TrpA1, TrpM8, TrpV1 and NaV1.7 are present in BN1 and BN2 but not detected in MEFs or in BAZ samples indicated by n.d. Expression is normalized to Gapdh. Expression levels set are relative to BN1 such that expression of BN1 =1.0. Bars and error bars represent means and SEMs from two independent biological replicates. (b) ΔF/Fo intensity plot showing the response of individual MEF derived BAZ induced neurons to 25mM KCl, 10 μM capsaicin (Cap), 100 μM Menthol (Men), and 100μM mustard oil (MO). Each cell is represented in each column. Cells that respond to KCl only (black circle), one Cap responsive cell is observed (red circle). (c) Representative calcium traces for 10μM capsaicin (Cap), 100 μM Menthol (Menth), and 100μM mustard oil (MO). Calcium transients were measured using Map2::GCAMP5.G. Calcium responses were calculated as the change in fluorescence (ΔF) over the initial fluorescence (Fo). Depolarization with 25 mM KCl was used at the beginning and end of each experiment to confirm neural identity and sustained functional capacity. (d and e) Change in fluorescent intensity of CoroNa-Green sodium dye with in individual iSNs following washes with KCl, KCl + Lidocaine, Menthol, and Capsaicin. (f) Representative images of iSNs (map2, red) with CoroNa-Green following washes with KCl, KCl + Lidocaine, Menthol, and Capsaicin

Supplementary Figure 8 iSN reprogramming does not require specialized embryonic precursors

BN1 and BN2 reprogramming does not require neural crest progenitors. (a) End-point RT-PCR for fibroblasts and neural crest markers. Minus RT (-RT) RNA heated to 70°C for 20 minutes and subsequently amplified with Gapdh primers. (b) FACS plots for p75/Thy1. A minor population of p75-positive cells are present in MEF preparations (c) Representative neural cells reprogrammed with BN1 from sorted MEFs that were negative for p75. (d) Reprogramming efficiency is not affected by removal of p75 positive cell population

Supplementary Figure 9 Expression of BN1 or BN2 reprograms human dermal fibroblasts to iSNs

(a) Expression of BN1 and BN2 converts human dermal fibroblasts to MAP2/TUJ1 double positive cells with neuronal morphologies. Images were taken 14 days after induction; dox was removed on day 8. Scale bar: 100 μm (b) Percent TUJ1 single positive (grey) and MAP2/TUJ1 double positive (green) cells generated from HEFs in BN1, BN2, and rtTA control conditions. (c) Neurons induced with BN1/2 express the peripheral sensory transcription factor ISL1 in the majority of MAP2/TUJ1 positive cells. (d) Neurons induced with BN1/2 express TRKA, TRKB, and TRKC. Scale bar: 25 µm. (e) Quantification of TUJ positive cells expressing TRKA, B, C. Error bars represent SEM

Supplementary Figure 10 Summary of electrophysiological data from human iSNs

(a) Representative images of iSNs with neural morphology attached to patch electrode. (b, c) Representative traces from BN2 iSNs showing the voltage response of multiple spiking cells (b) and single spiking cells (c), with single action potentials shown on the right. The input resistance of these iSNs is plotted as a function of the injected current in (d) and (f). The number of action potentials fired at increasing levels of current is shown in (e) and (g). (h) Physiological properties of combined BN1 and BN2 cells measured in whole-cell current clamp experiments. The cells are classified into single spiking and multiple spiking groups according to their response under suprathreshold levels of depolarizing current. Both groups have relatively high input resistance at resting membrane potential. The single spiking cells display significantly higher rheobase and spike threshold than the regular firing cells. The gain of the I-O curve and the spike amplitude is less for the single spiking cells than the regular firing types. Means and standard errors are shown (*: p<0.05; ***: p<0.005, two-tail t-test). Exact p-values: resting Vm (p = 0.69), input resistance (p = 0.3), rheobase (p = 0.003), I-O gain (p = 3×10−8), spike threshold (p = 0.0004), spike amplitude (p = 2×10−5), spike width (p = 0.05), capacitance (p = 0.9)

Supplementary Figure 11 The majority of iSNs exhibit calcium transients in response to depolarization

(a) Representative images of one field of view showing MAP2::GCAMP fluorescence intensity changes in human BN1 iSNs at two time points: before KCl addition and at the time of peak fluorescence intensity following KCl addition. (b) Image of BN1 iSNs in the same field of view as panel A after immunostaining with MAP2, TUJ1, and DAPI, showing that GCAMP fluorescent changes occur within MAP2/TUJ1 positive iSNs. (c) Calcium response curves from iSNs shown in panel A and B, calculated as the change in fluorescence (ΔF) over the initial fluorescence (Fo). (d) Quantification of the percentage of MAP2/TUJ1 positive cells that show a positive ΔF/Fo response to the addition of KCl

Supplementary Figure 12 Full-length gel images corresponding to cropped images in Figures 4a and 8a

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Blanchard, J., Eade, K., Szűcs, A. et al. Selective conversion of fibroblasts into peripheral sensory neurons. Nat Neurosci 18, 25–35 (2015). https://doi.org/10.1038/nn.3887

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