An In Vitro Model of Developmental Synaptogenesis Using Cocultures of Human Neural Progenitors and Cochlear Explants

Abstract

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

“…Recent literature supports the use of neural crest progenitors in a cell replacement therapy for deafness, given the molecular similarity of this population to placode-derived sensory neurons (Huisman and Rivolta, 2012;Nayagam et al, 2013). In addition, we have recently demonstrated that neurons derived from this induction protocol express key auditory neural proteins including NeuroD1, Brn3a, GATA3, Islet1 and neurofilament ( Fig.…”

“…A number of studies have now demonstrated the potential of stem cells to differentiate into appropriate neurosensory progenitors, including those of human origin (Shi et al, 2007;Chen et al, 2009Chen et al, , 2012Nayagam et al, 2013). The expression of key developmental markers in the differentiation of human stem cells toward an auditory neural lineage has recently been documented (Chen et al, 2012; and reviewed by Gunewardene et al, 2012) and includes the expression of key proteins and transcription factors Sox 2, Pax2/8, FoxG1, Six1, Nestin and Brn3a (Chen et al, 2012), Brn3a, GATA3 and peripherin (Shi et al, 2007), Pax2, Brn3a, peripherin, and neurofilament and NeuroD1, Brn3a, GATA3, Islet1, peripherin, and neurofilament (Gunewardene 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 (I Na ) and sustained outward K + currents (I K ).…”

Combined Growth Factor and Gene Therapy: An Approach for Hair Cell Regeneration and Hearing Recovery

“…In an in vitro study, neural precursors derived from hESCs were cultured along with cochlea in the 3D culture medium and it was observed that the precursor neurons were electrically active and they could differentiate into the sensory HC alongside the implanted cochlea. These results showed that neural precursors derived from hESCs might be useful in developing treatments that could directly replace the ANs [79].…”

Genes and Hearing Loss: Relationship to Oxidative Stress and Free Radical Formation

“…Although the strategies to date remain relatively ineffi cient in terms of cell yield, a number of laboratories have been successful in generating cells with molecular and functional phenotypes resembling sensory hairlike cells from ES cells and from terminally differentiated cells such as fi broblasts from mouse and human (e.g., Koehler et al 2013 ;Ouji et al 2013 ;Ronaghi et al 2014 ). Similar studies have been used to generate neuronal cells with characteristics of spiral ganglion neurons (e.g., Tong et al 2010 ;Nayagam et al 2012 ). Successful reprogramming of cells from humans carrying highly penetrant Mendelian mutations or lower penetrance risk variants should permit in-depth analysis of pathways associated with inner ear dysfunction in a cellular context that better approximates conditions in vivo.…”