Introduction Human pluripotent stem cells hPSCs i e human em

Introduction Human pluripotent stem h2 receptor antagonist (hPSCs), i.e., human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can give rise to functional neurons (reviewed in Gage and Temple, 2013). Human peripheral neurons are difficult to differentiate, but a recently published combinatorial small molecule-based approach was able to overcome this shortage (Chambers et al., 2012). These neurons express canonical markers of nociceptors, such as TAC1 (pro-peptide to Substance P), SCN9A (NAV1.7), and SCN10A (NAV1.8). They exhibit currents mediated by ASIC, GABAA receptors, HCN1, and KCNQ2/3 (Young et al., 2014). Neuropathic pain is a disabling and difficult to treat condition that cannot be adequately mimicked in animal models. Recently, inherited neuropathic pain syndromes were linked to mutations in voltage-gated sodium channels (NAVs) (Lampert et al., 2014), stressing the importance of TTXr NAVs in neuropathic pain and as targets for novel, specific pain treatment (Bagal et al., 2014). Nociceptive stimuli are conveyed by NAVs that comprise nine different subtypes expressed in human and rodents. Six are tetrodotoxin-sensitive (TTXs) (NAV1.1 to NAV1.4, NAV1.6, and NAV1.7), whereas three are resistant to TTX (TTXr) (NAV1.5, NAV1.8, and NAV1.9). Inherited small fiber neuropathies (SFN) are, among others, associated with mutations in NAV1.8 (Faber et al., 2012). NAVs undergo expression changes during development, and pain syndromes linked to NAV mutations occur at certain periods in life; while the NAV1.7-linked severe pain syndrome PEPD (paroxysmal extreme pain disorder) can start in utero, with symptoms declining during adulthood, the NAV1.7-linked erythromelalgia has its onset in adolescence, and SFN is observed in older adults (Lampert et al., 2014). This striking variation in disease onset may be due to modulation of the mutation phenotypes by co-expression of different NAV subtypes, but experimental support for this hypothesis is still missing. Expression and function of NAVs depend critically on the cellular background (Rush et al., 2006), sounding a strong note of caution when translating data from rodent to human. This holds particularly true for nociceptors whose excitability is controlled by a complex interplay between TTXs and TTXr NAVs. Thus, there is an urgent need for the availability of human nociceptors to investigate the pathophysiology and pharmacology of nociception in an appropriate model. Even though hPSC-derived nociceptors are a promising cellular model in pain research, a detailed investigation of their set of NAV subtypes is still missing. Using hPSC-derived nociceptors, generated by applying an optimized protocol (Chambers et al., 2012), we explored the maturational stages of hPSC-derived nociceptors and deciphered the electrophysiological characteristics of their TTXr NAVs. Our analysis revealed that the generated neurons not only express NAV1.8 and NAV1.9, but also the developmentally regulated NAV1.5, suggesting that the derived neurons represent a specific stage during development.
Results and Discussion
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Since the first derivation of mouse embryonic stem cells (mESCs) (Evans and Kaufman, 1981; Martin, 1981), several culture conditions have been developed for the maintenance of undifferentiated mESCs in vitro, including the use of serum-containing medium supplemented with leukemia inhibitory factor (LIF) (Smith et al., 1988; Williams et al., 1988) as well as serum-free N2B27 medium supplemented with LIF and bone morphogenetic protein 4 (BMP4) (Ying et al., 2003). LIF promotes mESC self-renewal by activating signal transducer and activator of transcription 3 (STAT3) (Niwa et al., 1998), while BMP4 can replace serum in supporting mESC self-renewal mediated by LIF/STAT3 (Ying et al., 2003). In 2008, we found that mESC self-renewal can be efficiently maintained by two small molecule inhibitors (2i), CHIR99021 and PD0325901 (CHIR and PD hereinafter) (Ying et al., 2008). CHIR and PD maintain self-renewal through inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK), respectively.