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PAIN 155 (2014) 1681–1682
www.elsevier.com/locate/pain
Commentary
Human pain in a dish: Native DRG neurons and differentiated pluripotent stem cells Sensitization of dorsal root ganglion (DRG) neurons caused by genetic mutations, nerve injury, or tissue inflammation underlies chronic pain [2,6,15]. Understanding the normal molecular basis of excitability of these neurons and how they respond to disease or injury is critical for the development of new and more effective drugs for treatment of pain. Mechanistic studies of pain pathophysiology have depended on rodents as tractable laboratory models to elucidate the role of specific molecules and circuits in the transduction and transmission of noxious stimuli along the afferent pain neuraxis and for the organismal response, and the conclusions have been extrapolated to humans. This approach depends on the implicit assumption that the basic mechanisms regulating the excitability of these neurons are conserved between experimental rodent models and humans. Although invaluable for testing pathophysiological bases for pain, the value of preclinical animal research has been questioned because of the poor translation of animal data into successful treatments in humans [10]. This has led to a growing interest in using cell-based assays, native DRG neurons, or sensory neurons differentiated from pluripotent stem cells (PSC), both to study molecular and cellular bases of noxious stimulus–evoked hyperexcitability and as cellular platforms to screen and validate novel drugs before embarking on costly clinical trials. DRG neurons have been used as an in vitro system to investigate mechanistic basis for spontaneous and stimulus-evoked excitability of nociceptors which underlie pain. For example, such assays have provided evidence confirming functional links of mutations in voltage-gated sodium channels in rare human pain disorders and more common painful peripheral neuropathy [6,7,9]. Although rodent DRG neurons were used in these assays, they were transfected with either wild-type or mutant human channel constructs, and the effect of the mutation on neuronal excitability is thus revealed. However, extrapolation from studies of native rodent DRG neurons to humans is more challenging because of notable differences between rodent and human DRG neurons [5,12,13]. A compelling example is provided by NaV1.9, a member of the voltage-gated sodium channels subfamily of ion channels that make up the electrogenisome that shapes action potential properties [14]. NaV1.9 current in native human DRG neurons manifests a 10 to 20 mV hyperpolarizing shift of activation compared to the NaV1.9 current in mouse and rat DRG neurons [5]. The marked enhancement of NaV1.9 activation in human DRG neurons is predicted to have profound effects on neuronal firing pattern, assuming that other components of the electrogenisome q
DOI of original article: http://dx.doi.org/10.1016/j.pain.2014.06.017
are regulated in a similar manner in rodents and humans. Whether the firing properties of human and rodent DRG neurons are quantitatively comparable remains to be established, and the study by Davidson et al. [4] in this issue is an important step in that direction. Davidson et al. have now described in some detail morphological and action potential properties of DRG neurons from 5 human donors with no overt pain before death. Although human DRG neurons have been studied [1,5,13], they typically involved limited number of studied neurons due to the paucity of viable human tissue for live cell analysis. The current study reported electrogenic properties of 141 small to medium DRG neurons (30–60 lm) and provided experimental evidence that a subset of these neurons responded to compounds that produce pain and itch, and that these neurons can be sensitized by proinflammatory mediators. They concluded that DRG neurons of this size range manifest properties of nociceptors and suggested that human DRG neurons may serve as an alternative to rodents as a preclinical platform for target validation. It is common for studies of this nature to leave many basic questions unanswered. Although the authors investigated firing properties of human DRG neurons, they did not provide data on the ionic conductances that underlie electrogenesis in these neurons. This study did not address the heterogeneity of the expression of known biomarkers in subpopulations of DRG neurons of different sensory modalities, which have been elegantly demonstrated by fate-mapping, immunohistochemical and functional studies in rodents; even discrimination between peptidergic and nonpeptidergic subpopulations of unmyelinated afferents could not be established because the IB4 binding epitope is missing in human versican. The data were not sufficiently robust to address variability among the donors, which may contribute to interindividual variability in response to noxious stimuli among humans. These and many other issues will have to be resolved in future studies. The data presented by Davidson et al., however, begin to establish a baseline and a benchmark for follow-up studies using native human DRG neurons and neurons differentiated from human PSC. The availability of DRG tissue to scientist through tissue banks and national tissue procurement organizations, or through individualized collaboration with medical centers with programs in place for organ procurement, will accelerate this line of investigation. Although successful differentiation of peripheral-type neurons from human embryonic stem cells or PSC, including a subpopulation with sensory neuronal properties, has been established [3,8,11], these studies did not provide comparison with native
http://dx.doi.org/10.1016/j.pain.2014.07.010 0304-3959/Ó 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1682
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Commentary / PAIN 155 (2014) 1681–1682
DRG neurons. Recently, a study reported a more in depth genomewide mRNA expression analysis during differentiation of sensory neurons from hPSC, and, importantly, compared the findings to data from native human DRG neurons [16]. Compared to native human DRG profile, Young et al. [16] reported the expression of 84% of ion channels by 30 days of differentiation of sensory neurons from hPSC. Although impressive for carrying out this comparative analysis, Young et al. limited the functional comparison to a subset of ion channels and did not investigate the rich heterogeneous sensory modalities of native human DRG neurons. The successful implementation of the 2 approaches—in vitro studies of human DRG neurons and induced hPSC differentiation into sensory neurons—hold the promise for qualitative and quantitative assessment of the expression of specific markers in human and rodent neurons, which is essential for a better understanding of the cellular basis of pain pathophysiology and novel drug development. Conflict of interest statement The author reports no conflict of interest. Acknowledgments SDH was funded by grants from the Rehabilitation Research Service and Medical Research Service, Department of Veterans Affairs. The Center for Neuroscience and Regeneration is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University. References [1] Anand U, Otto WR, Casula MA, Day NC, Davis JB, Bountra C, Birch R, Anand P. The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons. Neurosci Lett 2006;399:51–6. [2] Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 2009;139:267–84. [3] Chambers SM, Qi Y, Mica Y, Lee G, Zhang XJ, Niu L, Bilsland J, Cao L, Stevens E, Whiting P, Shi SH, Studer L. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 2012;30:715–20. [4] Davidson S, Copits BA, Zhang J, Page G, Ghetti A, Gereau RW IV. Human sensory neurons: Membrane properties and sensitization by inflammatory mediators. PAINÒ 2014;155:1861–70. [5] Dib-Hajj SD, Tyrrell L, Cummins TR, Black JA, Wood PM, Waxman SG. Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons. FEBS Lett 1999;462:117–20.
[6] Dib-Hajj SD, Yang Y, Black JA, Waxman SG. The NaV1.7 sodium channel: from molecule to man. Nat Rev Neurosci 2013;14:49–62. [7] Faber CG, Lauria G, Merkies IS, Cheng X, Han C, Ahn HS, Persson AK, Hoeijmakers JG, Gerrits MM, Pierro T, Lombardi R, Kapetis D, Dib-Hajj SD, Waxman SG. Gain-of-function NaV1.8 mutations in painful neuropathy. Proc Natl Acad Sci USA 2012;109:19444–9. [8] Guo X, Spradling S, Stancescu M, Lambert S, Hickman JJ. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials 2013;34:4418–27. [9] Huang J, Han C, Estacion M, Vasylyev D, Hoeijmakers JG, Gerrits MM, Tyrrell L, Lauria G, Faber CG, Dib-Hajj SD, Merkies IS, Waxman SG. Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy. Brain 2014;137:1627–42. [10] Mogil JS. Animal models of pain: progress and challenges. Nat Rev Neurosci 2009;10:283–94. [11] Pomp O, Brokhman I, Ziegler L, Almog M, Korngreen A, Tavian M, Goldstein RS. PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Res 2008;1230:50–60. [12] Serrano A, Mo G, Grant R, Pare M, O’Donnell D, Yu XH, Tomaszewski MJ, Perkins MN, Seguela P, Cao CQ. Differential expression and pharmacology of native P2X receptors in rat and primate sensory neurons. J Neurosci 2012;32:11890–6. [13] Valeyev AY, Hackman JC, Wood PM, Davidoff RA. Pharmacologically novel GABA receptor in human dorsal root ganglion neurons. J Neurophysiol 1996;76:3555–8. [14] Waxman SG. Sodium channels, the electrogenisome, and the electrogenistat: lessons and questions from the clinic. J Physiol 2012;590:2601–12. [15] Woolf CJ. Pain: morphine, metabolites, mambas, and mutations. Lancet Neurol 2013;12:18–20. [16] Young GT, Gutteridge A, Fox HD, Wilbrey AL, Cao L, Cho LT, Brown AR, Benn CL, Kammonen LR, Friedman JH, Bictash M, Whiting P, Bilsland JG, Stevens EB. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research. Mol Ther 2014. http://dx.doi.org/10.1038/mt.2014.86, [Epub ahead of print].
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Sulayman D. Dib-Hajj Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA Rehabilitation Research Center, Veterans Administration Connecticut Healthcare System, West Haven, CT 06516, USA ⇑ Address: Center for Neuroscience and Regeneration Research, 127A, Bldg 34, VA Connecticut Healthcare System, 950 Campbell Ave, West Haven, CT 06516, USA. Tel.: +1 (203) 937 3802; fax: +1 (203) 937 3801. E-mail address: [email protected]
PAIN 155 (2014) 1681–1682
www.elsevier.com/locate/pain
Commentary
Human pain in a dish: Native DRG neurons and differentiated pluripotent stem cells Sensitization of dorsal root ganglion (DRG) neurons caused by genetic mutations, nerve injury, or tissue inflammation underlies chronic pain [2,6,15]. Understanding the normal molecular basis of excitability of these neurons and how they respond to disease or injury is critical for the development of new and more effective drugs for treatment of pain. Mechanistic studies of pain pathophysiology have depended on rodents as tractable laboratory models to elucidate the role of specific molecules and circuits in the transduction and transmission of noxious stimuli along the afferent pain neuraxis and for the organismal response, and the conclusions have been extrapolated to humans. This approach depends on the implicit assumption that the basic mechanisms regulating the excitability of these neurons are conserved between experimental rodent models and humans. Although invaluable for testing pathophysiological bases for pain, the value of preclinical animal research has been questioned because of the poor translation of animal data into successful treatments in humans [10]. This has led to a growing interest in using cell-based assays, native DRG neurons, or sensory neurons differentiated from pluripotent stem cells (PSC), both to study molecular and cellular bases of noxious stimulus–evoked hyperexcitability and as cellular platforms to screen and validate novel drugs before embarking on costly clinical trials. DRG neurons have been used as an in vitro system to investigate mechanistic basis for spontaneous and stimulus-evoked excitability of nociceptors which underlie pain. For example, such assays have provided evidence confirming functional links of mutations in voltage-gated sodium channels in rare human pain disorders and more common painful peripheral neuropathy [6,7,9]. Although rodent DRG neurons were used in these assays, they were transfected with either wild-type or mutant human channel constructs, and the effect of the mutation on neuronal excitability is thus revealed. However, extrapolation from studies of native rodent DRG neurons to humans is more challenging because of notable differences between rodent and human DRG neurons [5,12,13]. A compelling example is provided by NaV1.9, a member of the voltage-gated sodium channels subfamily of ion channels that make up the electrogenisome that shapes action potential properties [14]. NaV1.9 current in native human DRG neurons manifests a 10 to 20 mV hyperpolarizing shift of activation compared to the NaV1.9 current in mouse and rat DRG neurons [5]. The marked enhancement of NaV1.9 activation in human DRG neurons is predicted to have profound effects on neuronal firing pattern, assuming that other components of the electrogenisome q
DOI of original article: http://dx.doi.org/10.1016/j.pain.2014.06.017
are regulated in a similar manner in rodents and humans. Whether the firing properties of human and rodent DRG neurons are quantitatively comparable remains to be established, and the study by Davidson et al. [4] in this issue is an important step in that direction. Davidson et al. have now described in some detail morphological and action potential properties of DRG neurons from 5 human donors with no overt pain before death. Although human DRG neurons have been studied [1,5,13], they typically involved limited number of studied neurons due to the paucity of viable human tissue for live cell analysis. The current study reported electrogenic properties of 141 small to medium DRG neurons (30–60 lm) and provided experimental evidence that a subset of these neurons responded to compounds that produce pain and itch, and that these neurons can be sensitized by proinflammatory mediators. They concluded that DRG neurons of this size range manifest properties of nociceptors and suggested that human DRG neurons may serve as an alternative to rodents as a preclinical platform for target validation. It is common for studies of this nature to leave many basic questions unanswered. Although the authors investigated firing properties of human DRG neurons, they did not provide data on the ionic conductances that underlie electrogenesis in these neurons. This study did not address the heterogeneity of the expression of known biomarkers in subpopulations of DRG neurons of different sensory modalities, which have been elegantly demonstrated by fate-mapping, immunohistochemical and functional studies in rodents; even discrimination between peptidergic and nonpeptidergic subpopulations of unmyelinated afferents could not be established because the IB4 binding epitope is missing in human versican. The data were not sufficiently robust to address variability among the donors, which may contribute to interindividual variability in response to noxious stimuli among humans. These and many other issues will have to be resolved in future studies. The data presented by Davidson et al., however, begin to establish a baseline and a benchmark for follow-up studies using native human DRG neurons and neurons differentiated from human PSC. The availability of DRG tissue to scientist through tissue banks and national tissue procurement organizations, or through individualized collaboration with medical centers with programs in place for organ procurement, will accelerate this line of investigation. Although successful differentiation of peripheral-type neurons from human embryonic stem cells or PSC, including a subpopulation with sensory neuronal properties, has been established [3,8,11], these studies did not provide comparison with native
http://dx.doi.org/10.1016/j.pain.2014.07.010 0304-3959/Ó 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1682
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Commentary / PAIN 155 (2014) 1681–1682
DRG neurons. Recently, a study reported a more in depth genomewide mRNA expression analysis during differentiation of sensory neurons from hPSC, and, importantly, compared the findings to data from native human DRG neurons [16]. Compared to native human DRG profile, Young et al. [16] reported the expression of 84% of ion channels by 30 days of differentiation of sensory neurons from hPSC. Although impressive for carrying out this comparative analysis, Young et al. limited the functional comparison to a subset of ion channels and did not investigate the rich heterogeneous sensory modalities of native human DRG neurons. The successful implementation of the 2 approaches—in vitro studies of human DRG neurons and induced hPSC differentiation into sensory neurons—hold the promise for qualitative and quantitative assessment of the expression of specific markers in human and rodent neurons, which is essential for a better understanding of the cellular basis of pain pathophysiology and novel drug development. Conflict of interest statement The author reports no conflict of interest. Acknowledgments SDH was funded by grants from the Rehabilitation Research Service and Medical Research Service, Department of Veterans Affairs. The Center for Neuroscience and Regeneration is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University. References [1] Anand U, Otto WR, Casula MA, Day NC, Davis JB, Bountra C, Birch R, Anand P. The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons. Neurosci Lett 2006;399:51–6. [2] Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 2009;139:267–84. [3] Chambers SM, Qi Y, Mica Y, Lee G, Zhang XJ, Niu L, Bilsland J, Cao L, Stevens E, Whiting P, Shi SH, Studer L. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 2012;30:715–20. [4] Davidson S, Copits BA, Zhang J, Page G, Ghetti A, Gereau RW IV. Human sensory neurons: Membrane properties and sensitization by inflammatory mediators. PAINÒ 2014;155:1861–70. [5] Dib-Hajj SD, Tyrrell L, Cummins TR, Black JA, Wood PM, Waxman SG. Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons. FEBS Lett 1999;462:117–20.
[6] Dib-Hajj SD, Yang Y, Black JA, Waxman SG. The NaV1.7 sodium channel: from molecule to man. Nat Rev Neurosci 2013;14:49–62. [7] Faber CG, Lauria G, Merkies IS, Cheng X, Han C, Ahn HS, Persson AK, Hoeijmakers JG, Gerrits MM, Pierro T, Lombardi R, Kapetis D, Dib-Hajj SD, Waxman SG. Gain-of-function NaV1.8 mutations in painful neuropathy. Proc Natl Acad Sci USA 2012;109:19444–9. [8] Guo X, Spradling S, Stancescu M, Lambert S, Hickman JJ. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials 2013;34:4418–27. [9] Huang J, Han C, Estacion M, Vasylyev D, Hoeijmakers JG, Gerrits MM, Tyrrell L, Lauria G, Faber CG, Dib-Hajj SD, Merkies IS, Waxman SG. Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy. Brain 2014;137:1627–42. [10] Mogil JS. Animal models of pain: progress and challenges. Nat Rev Neurosci 2009;10:283–94. [11] Pomp O, Brokhman I, Ziegler L, Almog M, Korngreen A, Tavian M, Goldstein RS. PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Res 2008;1230:50–60. [12] Serrano A, Mo G, Grant R, Pare M, O’Donnell D, Yu XH, Tomaszewski MJ, Perkins MN, Seguela P, Cao CQ. Differential expression and pharmacology of native P2X receptors in rat and primate sensory neurons. J Neurosci 2012;32:11890–6. [13] Valeyev AY, Hackman JC, Wood PM, Davidoff RA. Pharmacologically novel GABA receptor in human dorsal root ganglion neurons. J Neurophysiol 1996;76:3555–8. [14] Waxman SG. Sodium channels, the electrogenisome, and the electrogenistat: lessons and questions from the clinic. J Physiol 2012;590:2601–12. [15] Woolf CJ. Pain: morphine, metabolites, mambas, and mutations. Lancet Neurol 2013;12:18–20. [16] Young GT, Gutteridge A, Fox HD, Wilbrey AL, Cao L, Cho LT, Brown AR, Benn CL, Kammonen LR, Friedman JH, Bictash M, Whiting P, Bilsland JG, Stevens EB. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research. Mol Ther 2014. http://dx.doi.org/10.1038/mt.2014.86, [Epub ahead of print].
⇑
Sulayman D. Dib-Hajj Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA Rehabilitation Research Center, Veterans Administration Connecticut Healthcare System, West Haven, CT 06516, USA ⇑ Address: Center for Neuroscience and Regeneration Research, 127A, Bldg 34, VA Connecticut Healthcare System, 950 Campbell Ave, West Haven, CT 06516, USA. Tel.: +1 (203) 937 3802; fax: +1 (203) 937 3801. E-mail address: [email protected]
