An important challenge to overcome

An important challenge to overcome in developing a cell replacement therapy for hearing loss is the development of a functionally stable stem cell-derived neural population (Needham et al., 2013). This entails both the development of electrically active neurons and their functional integration including formation of synapses with target neurons in the cochlear nucleus. We and others have previously shown that human stem cell-derived neurons can fire action potentials, and possess the core currents and channel families necessary for this task (Chen et al., 2009; Nayagam et al., 2013). Among these are the inward Na+ currents (INa) and sustained outward K+ currents (IK). These are arguably the most basic currents necessary to instigate action potentials, and therefore communicate meaningful signals to their target/s. The next milestone in our experimentation is to develop neurons with an electrical phenotype capable of processing information in a similar manner to the primary auditory neurons. Most notably, the glutamatergic primary auditory neurons possess a large complement of ion muscarinic receptor antagonists that enable them to respond to complex signals with temporal precision (reviewed Needham et al., 2013). A key feature of this neural phenotype is the ability to reliably follow high frequency stimulation since this is a hallmark of acoustic stimuli, as well as the electrically encoded input from a cochlear implant. Interestingly, little is known about the definitive firing rates of auditory neurons in response to electrical stimulation in humans. However, what is clear from clinical studies, is that pitch discrimination deteriorates as stimulation levels approach 300 pulses per second (Shannon, 1983; Zeng, 2002; Vandali et al., 2013). Thus, based upon these data, it seems reasonable to expect that replacement neurons be capable of firing at similar rates to endogenous auditory neurons as a reduction in firing entrainment would likely affect the amount of information encoded in the signal relayed to the brain, and therefore the accurate perception of sound. Here we examine the electrical profile of human embryonic stem cell (hESC) derived neurosensory progenitors over time in vitro, and compare their responses to high frequency stimulation with that of the primary auditory neuron population.
Materials and methods
Results
Discussion This study describes the activity of human stem cell-derived neurons after extended time in culture, and in response to high frequency stimulation. The findings reported herein, confirm that our differentiation assay consistently produces a population of electrically active neurons which possess Na+ and K+ currents. We have observed that the described population of stem cell-derived neurons predominantly displays a rapidly adapting phenotype. Classified in accordance with the previously defined convention for primary auditory neurons (Mo and Davis, 1997a; Adamson et al., 2002b), rapidly adapting cells fire between one and six action potentials during a 300ms stimulus, while slowly adapting cells fire seven or more action potentials over the same period. Interestingly, the predominance of this phenotype is comparable with recordings from early postnatal primary auditory neurons in vitro, where the majority displayed a rapidly adapting profile (Needham et al., 2012). In addition, murine stem cell-derived neurons uniformly show rapidly adapting responses over a period of 12days in vitro (Tong et al., 2010). The rapidly adapting phenotype identified in the current study was stable for 5weeks in culture, supporting the use of our excitable population of cells as replacement cells for auditory neurons. The firing profile and physiological properties recorded from this population of stem cell-derived neurons nicely match those reported for embryonic day 14–15 (E14–15) mammalian auditory neurons in situ (Marrs and Spirou, 2012). In particular, both populations exhibit similar RMP, RIN, and firing threshold, and display a broader action potential when compared to postnatal auditory neurons. Of additional interest is the absence of a voltage ‘sag’ in both E14–15 auditory neurons and our population of stem cell-derived neurons. The presence of a ‘sag’, and the hyperpolarization-activated current (Ih) which underlies this activity, is consistently reported in postnatal primary auditory neurons in vitro (Mo and Davis, 1997b; Szabo et al., 2002; Zhou et al., 2005; Needham et al., 2012), and has also been noted in later embryonic development of both primary auditory neurons and auditory brainstem neurons (Marrs and Spirou, 2012). This hints at Ih and voltage ‘sag’ as key markers of auditory neuron maturity. Notably, Ih, which regulates neuronal excitability in part through control of RIN, can be influenced by the application of neurotrophins (Needham et al., 2012). Likewise, a lower (i.e. more hyperpolarized) RMP, which in our stem cell-derived neurons remained in the order of −50mV across the 5weeks in vitro, is also suggestive of a maturing neural phenotype and may also be assisted by the presence of BDNF in the culture media (Purcell et al., 2013). As recently demonstrated by Purcell et al. (2013), expression of the K+ channel KCNQ4 dramatically increased in stem cell-derived neurons following BDNF exposure. These authors also reported a concomitant reduction in RMP, thereby highlighting the potential to produce specific neuronal subtypes using appropriate soluble molecules.