Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

Abstract

In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

Directing Human Induced Pluripotent Stem Cells into a Neurosensory Lineage for Auditory Neuron Replacement

“…The stem cell–derived neurospheres and cultures of primary ANs (prepared from P4-P7 Wistar rat pups, as previously described 46,50 ) were plated onto glass coverslips [10 mm diameter, Menzel-Glaser; precoated with polyornithine (500 μg/mL; Sigma-Aldrich) and mouse laminin (0.01 mg/mL; Invitrogen)] and grown in vitro at 37°C, 10% CO 2 . As previously described, 44,46 coverslips were transferred to a recording chamber fitted to an AxioExaminer D1 microscope (Carl Zeiss) for electrophysiological recordings.…”

“…44 Importantly, we have recently reported the use of an induction protocol for generating a mixed population of neurosensory progenitors from hESCs. 44 Sensory neurons derived from this protocol exhibited electrophysiological properties akin to embryonic mammalian ANs 45,46 and made synapses on appropriate peripheral targets in vitro . 44 …”

“…44,46,47 Given their close association during development, it is possible that the neurons derived from this heterogeneous sphere-forming assay contain auditory-like precursors. 12 This study therefore aimed to use this established protocol to determine if hiPSCs could differentiate into a neurosensory-like lineage in a similar manner to that previously reported using hESCs.…”

“…12 This study therefore aimed to use this established protocol to determine if hiPSCs could differentiate into a neurosensory-like lineage in a similar manner to that previously reported using hESCs. 44,46 We chose to characterize the potential of two hiPSC lines to differentiate into a neurosensory lineage, using hESCs as a control. To our knowledge, this is the first study reporting the potential for hiPSCs to differentiate toward an auditory neuron-like lineage.…”

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model

“…These neural networks gradually break down over a long period in XP-A patients. Differentiation methods for sensory neurons from hESCs have been reported (Needham et al, 2014;Shi et al, 2007). These hESC-derived neurons were able to form synapses with hair cells in cultured sensory epithelia from the inner ears of mice or rats in vitro (Nayagam et al, 2013).…”