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Sensory neurons

The sense in reprogramming

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The overexpression of certain transcription factors in fibroblasts has the potential to rapidly generate various classes of neurons in vitro, which could prove beneficial for mechanistic studies of disease and drug screening. Two new studies now show that mouse and human fibroblasts can be reprogrammed to become peripheral sensory neurons through this approach.

Peripheral sensory neurons are derived from precursors that express the transcription factors neurogenin 1 (NGN1) and/or NGN2. These proteins are not expressed in mature sensory neurons, which instead have been shown to express the transcription factor BRN3A (also known as POU4F1).

Baldwin and colleagues transiently expressed BRN3A and NGN1 or BRN3A and NGN2 in mouse and human embryonic fibroblasts to see whether they could generate cells with the characteristic features of sensory neurons. Following the expression of either combination of factors, most of the mouse and human cells showed a neuron-like morphology. In addition, the large majority of these cells expressed multiple neuronal markers and exhibited electrophysiological properties consistent with functional neurons, indicating that they could be considered induced neurons.

Nociceptors and puritoceptors, mechanoceptors, and proprioceptors — the three functional subgroups of peripheral sensory neurons — selectively express different types of tropomyosin-related kinase (TRKA, TRKB and TRKC, respectively). Interestingly, most of the mouse and human induced neurons expressed one of these three receptors. Moreover, calcium assays revealed subpopulations of the mouse and human cells that were selectively responsive to known pain- and itch-inducing ligands. Thus, these data indicate that functional sensory neurons can be generated in vitro from embryonic fibroblasts through the expression of two transcription factors.

In their study, Woolf and colleagues aimed to specifically generate nociceptors (as opposed to sensory neurons more broadly) from fibroblasts. To do so, they first generated mice in which expression of the reporter tdTomato was dependent on expression of TRPV1, a cation channel that is found in nociceptors and responds to caspaicin and noxious heat. They isolated embryonic fibroblasts from this line and expressed different combinations of transcription factors in these cells to induce tdTomato expression.

This screening approach revealed that the simultaneous expression of five transcription factors — ASC1, MYT1L, NGN1, ISL2 and KLF7 — could induce the expression of tdTomato and hence TRPV1. Immunohistochemistry and RT-PCR analyses revealed that these cells also expressed neuronal and other nociceptor markers. Moreover, caspaicin activated 40% of these cells, as revealed by calcium imaging and extracellular recordings. Together, these data indicate that the fibroblasts were reprogrammed to generate functional nociceptors.

Peripheral sensitization of nociceptors can underlie low-threshold clinical pain hypersensitivity. Interestingly, pretreatment of tdTomato-expressing cells with prostaglandin E2, which sensitizes TRPV1, increased the responsiveness of these cells to low doses of caspaicin, suggesting that induced nociceptors can be used to model inflammatory sensitization.

Finally, the authors simultaneously expressed the five identified transcription factors in human fibroblasts and were again able to generate cells that expressed neuronal markers and that had electrophysiological properties resembling those of nociceptors. Furthermore, they were able to generate nociceptors from fibroblasts taken from individuals with familial dysautonomia. These neurons show impaired neurite growth and a reduced number of branches per cell, compared with induced nociceptors from healthy controls, indicating that induced nociceptors may provide insights into disease pathophysiology.

these studies reveal techniques for generating functional peripheral sensory neurons from mouse and human fibroblasts in vitro

Together, these studies reveal techniques for generating functional peripheral sensory neurons from mouse and human fibroblasts in vitro and highlight the potential for use of such cells in the modelling of pathological conditions.

References

  1. Blanchard, J. W. et al. Selective conversion of fibroblasts into peripheral sensory neurons. Nature Neurosci. http://dx.doi.org/10.1038/nn.3887 (2014)

  2. Wainger, B. J. et al. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nature Neurosci. http://dx.doi.org/10.1038/nn.3886 (2014)

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Yates, D. The sense in reprogramming. Nat Rev Neurosci 16, 1 (2015). https://doi.org/10.1038/nrn3894

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  • DOI: https://doi.org/10.1038/nrn3894

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