When considering the implantation of exogenous cells into the cochlea, a wide range of factors must be taken into account that have an important effect on the fate of the implanted cells and ultimately determine treatment outcome. Considerations such as the type of exogenous cells, their differentiation status and potential to differentiate towards other cell types, the selected route of implantation and the host micro-environment all play key roles in the survival and integration of the implanted cell population within the host tissue (Figure 1).
Figure 1 Factors to be considered regarding the use of exogenous cells to replace damaged cochlear cell types.
The ultimate fate of implanted exogenous cells, regarding their survival, differentiation towards desired cell phenotypes, their migration to relevant locations in the cochlea and central auditory targets, and their physical and functional integration within the host tissue, strongly depends on a variety of factors such as the type of donor cell, its differentiation status and differentiation potential, the route of implantation and the host micro-environment, among others.
Tissue-specific progenitor cells
There are numerous studies reporting on the potential of different exogenous cell types to replace sensory cells in the damaged inner ear. Some of the work has focused on cells that, although coming from other tissues, may still share some relevant characteristics with otic cell types. Wei et al demonstrated the capacity of ciliated ependymal cells, obtained from the forebrain germinal zone of adult mice, to get incorporated into streptomycin-treated cochlear explants, express markers typical of HCs and establish functional synapses with primary auditory neurons. In addition, the authors presented data indicating that neural stem cells (NSCs) isolated from the subventricular zone could be differentiated in vitro to neurons that established functional contacts with denervated HCs and with adult SGNs in corresponding co-cultures. In vivo work carried out by Hu et al demonstrated significant migration of adult mouse NSCs transplanted into adult guinea pig cochleae to relevant locations such as the organ of Corti (OC), the spiral ganglion and the auditory nerve tract. However, NSC survival rates were very low; neomycin-induced damage to the cochlea and also Neurogenin2-transduction of the NSCs prior to transplantation improved differentiation of the transplanted NSCs towards a neuronal phenotype and increased NSC survival. Higher rates of cell survival were registered by Regala et al following transplantation of murine adult NSCs into surgically damaged vestibulocochlear nerves in adult rats; importantly, the authors observed migration of murine cells to the brain stem in 50% of the transplanted animals. In another series of experiments, Edin et al obtained SGN-like cells from human neural progenitors (NPs) by applying culture conditions used to maintain primary cultures of guinea pig and human SGNs; the cells developed morphological features and expressed an array of markers typical of SGNs.
Although NSCs from the subventricular zone could be used in autografts, thus overriding the need for immunosuppression, their isolation poses a series of technical issues. Therefore, other alternatives have been studied, such as the use of precursor cells that are resident in the olfactory epithelium[10,11], a readily accessible tissue. These precursors exhibit stem cell-like properties and express markers typical of otic HCs (MYOSIN VIIA, CALRETININ, ESPIN, PRESTIN) when co-cultured with cells from adult cochlea or when exposed to cochlea-conditioned medium. Promising results were obtained by Xu et al, when implanting olfactory epithelium NSCs into the scala tympani of a rat model of noise-induced hearing loss; the implanted NSCs survived and migrated towards host spiral ganglion neurons, although they did not reach the OC. Yet the authors registered improved ABR results in NSC-treated deafened animals, compared to those from non-implanted deafened controls.
Notwithstanding, inner ear progenitors have been isolated from embryonic[13,14] and adult human tissues that would be highly suitable donor cells; however, this is not a likely option, given their very low numbers. Interestingly, Stefan Heller and his group have shown that it is possible to isolate cochlear stem cells from post-mortem tissues without loss of their self-renewing and differentiation potential during the first 10 days following death.
Mesenchymal stem cells
Mesenchymal stem cells (MSCs) constitute another candidate donor cell source[17-21]. These are readily accessible cells with potential to differentiate into multiple lineages, which makes them highly desirable candidates for autologous cell-based therapies; importantly, they exert immunosuppressive properties and it has been recently shown that transtympanic MSC administration into immunocompetent rat hosts does not elicit an inflammatory response. Kondo et al demonstrated the potential of murine MSCs (mMSCs) to acquire features of post-mitotic neurons following exposure to sonic hedgehog and retinoic acid (RA); the cells expressed a whole range of glutamatergic sensory neuron markers (SOX10, GATA3, GLUR4, VGLUT1, CALRETININ). An additional soluble protein present in hindbrain/somite/otocyst-conditioned medium and also in embryonic day 18 (E18) OC was required to induce the expression of additional sensory neuron markers (Brn3a, Neurogenin1, NeuroD). Further work by this group demonstrated that Wnt signalling induces the expression of a whole array of sensory neuron markers through the up-regulation of T cell leukemia 3 (Tlx3), a transcription factor that promotes differentiation towards glutamatergic phenotypes. Infusion of Wnt1 was shown to increase the survival and engraftment rates of mMSCs implanted into the modiolus of ouabain-treated Mongolian gerbils. Moreover, the implanted cells migrated throughout all the cochlear turns and reached the spiral ganglion; some of the cells adopted a clearly neuronal morphology and expressed neuronal markers.
In another series of experiments, Jeon et al demonstrated that mMSCs could be differentiated to HC-like cells by exposing the mMSCs to a culture regime used to differentiate mouse embryonic stem cells (mESCs) into HC-like cells and then over-expressing Math1, a well-known HC master gene. mMSCs were differentiated to neurosensory progenitors (mMSC-NsPs) that expressed markers of early otic development, such as Otx2, Nestin, Sox2, Musashi, the early HC genes Math1, Brn3c and GATA3, and sensory neuronal markers such as TrkB and TrkC, among others. Transfection of mMSC-NsPs with Math1 induced expression of the HC genes MyosinVIIa and Espin, and other supporting cell (SC) and neuronal markers. Differentiation to a HC phenotype was promoted by co-culturing non-transfected mMSC-NsPs with E13 chick otocyst cells and also by injecting these cells into chick otocysts; of note, the injected cells integrated into chick otic epithelia, especially at sites of damage. Interestingly, when similar studies to those carried out by Kondo et al and Jeon et al were conducted on human MSCs (hMSCs), some differences were observed in the response of these human cells to differentiation cues, compared to that of their murine counterparts[19,20]. Therefore, Durán-Alonso et al applied to hMSC cultures protocols that had been employed to direct human foetal auditory stem cells (hFASCs) from the cochleae of 9-11-week-old foetuses towards HC and auditory neuron fates. hMSCs were initially differentiated to neural progenitors (NPs); further treatment of hMSC-NPs resulted in induced expression of combinations of HC or SGN markers, depending on the culture regimes being applied. HC marker expression (ATOH1, MYOSINVIIa, BRN3C, CALRETININ) was only observed following treatment of hMSC-NPs that had been generated in suspension cultures, pointing to differences in the NP populations that had been obtained in floating and in adherent cultures. Unlike the results obtained by Jeon et al on mMSCs, co-culture of hMSC-NPs with chick otocyst cells did not promote differentiation towards the HC lineage. Expression of sensory neuron markers (SOX2, GATA3, NGN1, ISLET1, NF200) could only be induced in hMSC-NP cultures that had undergone RA treatment, thought to render the cultures responsive to subsequent differentiation cues, such as bFGF. Additional experiments demonstrated that co-culture of 3D hMSC-NPs with murine cochlear explants promoted the expression of the neuronal marker NF-200 in these cells. Differently from the protocols applied by Durán-Alonso et al, based on the use of defined media containing specific growth factor combinations, Boddy et al exposed hMSC cultures to media conditioned by hFASCs and observed sequential up-regulation of otic progenitor (PAX8, PAX2, SOX2), HC (ATOH1, BRN3C, MYOSINVIIA) and sensory neuron (NGN1, BRN3A) markers over time. A role was demonstrated for Wnt signalling at the early stages of otic induction. Additional work by Bas et al demonstrated the capacity of human nasal MSCs to integrate into gentamicin-treated cochlear explants from post-natal rats, mostly in the spiral ganglion region; MSCs did not integrate into undamaged explants. Higher numbers of cells expressing βIII-TUBULIN were observed in cultures of damaged cochleae that had received hMSCs compared to those that had not been cultured with the human cells; over half of these neurons were hMSC-derived, indicating both differentiation of the exogenous cells and a protective effect on remaining SGNs. hMSC-derived neurons were excitable, and projected neurites towards the sensory epithelium, further promoted by Wnt signalling activation. In a different set of experiments, Schäck et al explored the possibility to direct the differentiation of hMSCs to a glutamatergic neuron phenotype by conditionally expressing Ngn1, as already demonstrated by Reyes et al on mESCs. hMSCs were refractory to adopting the desired fate as, although some glutamatergic neuronal markers were induced over time, their expression was not maintained once conditional expression of Ngn1 was halted.
ESCs and induced pluripotent stem cells
In vivo survival and differentiation of transplanted stem cell types: Not-withstanding the valuable data obtained on the various types of exogenous cells mentioned above, the main advancements in the field have come from exploiting the great proliferative and multilineage differentiation potential offered by ESCs and induced pluripotent stem cells (iPSCs)[1,5,31]. Experiments have been carried out to investigate the influence of the host environment on the survival and differentiation of transplanted stem cells. Survival and induction of neuronal marker expression have been demonstrated at various timepoints following implantation into various in vivo animal models[8,32,33]. Additionally, some of these cells were seen to migrate to relevant locations such as the brain stem[8,32,33]; of note, work by Zhu et al reported teratoma formation in a number of recipient cochleae following transplantation of murine iPSCs (miPSCs). Genetic modification of donor stem cells prior to their implantation in order to favour their in vivo survival and/or differentiation has also been carried out. An example of this is the work carried out by Reyes et al; transient Ngn1 expression in mECSs following their implantation into the scala tympani of kanamycin-treated guinea pigs resulted in increased migration and neuronal differentiation rates, compared to those of mESC controls.
In vitro differentiation. Effect of the otic micro-environment on differentiating cell types: Another line of work has pursued the in vitro differentiation of ESCs and iPSCs towards inner ear sensory cell types. A major breakthrough came from the observations by Stefan Heller’s group that sequential incubation of mESC-derived embryoid bodies in serum-free medium (SFM) containing combinations of EGF, IGF-1 and bFGF resulted in the emergence of inner ear progenitor cells in the cultures; cells expressed markers that are seen during otic vesicle formation, such as Nestin, Otx2, Pax2, Bmp7 and Jagged1. In order to promote further maturation of these progenitor cells, the cultures were maintained in defined medium following withdrawal of growth factors; early markers Nestin, Pax2 and Bmp7 were downregulated, while the expression of HC genes such as Math1, Pou4f3, Jagged1, Myosin VIIa, Parvalbumin, AchRα9, p31Kip1 was induced; Espin was also expressed, indicative of stereociliary morphogenesis. Timing of expression and co-expression patterns of the various genes supported the hypothesis that cultures were mimicking in vivo inner ear developmental stages. Importantly, the authors demonstrated integration of inner ear progenitors into developing chick otic epithelia. Integration preferentially occurred in areas of the epithelium that had been damaged during surgery; progenitor cells that incorporated within HC-bearing regions up-regulated the HC marker MYOSIN VIIA and some developed F-actin-rich hair bundles that were labelled with an anti-ESPIN antibody, demonstrating an instructive role of the otic environment. The same group later developed a more elaborate step-wise approach to differentiate mESCs and miPSCs, where ectodermal induction was promoted at the expense of endoderm and mesoderm, and the formation of anterior ectoderm was favoured by the addition of IGF1; FGF combinations were then applied as the main otic inductive signals. Removal of the growth factors present in the medium resulted in Math1 and MyosinVIIa expression; however , the cells did not present the typical HC morphology nor were hair bundle markers such as Espin detected. These features were only observed when the otic progenitor cells were grown on a layer of mitotically inactivated E18 chicken utricle stromal cells; the cells in these cultures exhibited stereociliary bundles and responded to mechanical stimulation in similar ways to those of immature HCs. Heller’s group then extended their studies to hESCs, applying to their cultures a modification of the treatment regime used for mESCs and miPSCs. The number of cells shown to express a combination of various HC markers was low and the cells resembled nascent HCs that did not further mature by increasing culture times but died instead. Additional studies were carried out on monolayer cultures of hESCs and hiPSCs, in an attempt to better characterize the conditions required to obtain bona fide otic cell types from these cultures; this work identified retinoic acid as a critical factor for bFGF-induced expression of early otic markers in pre-placodal ectoderm cells. Nevertheless, no further differentiation of the cells was attained, indicating that the monolayer culture model lacked some of the factors found in aggregate cultures that promote the differentiation of otic progenitor cells. Supporting these findings, Abboud et al obtained better results when applying an otic induction protocol [modified from (34)] to mESC cultures grown in floating conditions compared to cultures grown as monolayers. A greater proportion of the cells grown under non-adherent conditions expressed otic progenitor (PAX2, SIX1, EYA1, SOX2) and early HC markers (MYOSIN VIIA, POU4F3) following treatment, compared to adherent cultures. Following induction, floating cultures were partially dissociated and grafted into neomycin-damaged murine cochlear explants; a small number of these cells survived and integrated into the host tissue, preferentially in damaged areas of the OC, and expressed MYOSIN VIIA. Interestingly, this was not observed for any of the progenitors that had integrated outside the lesioned area. Notwithstanding, attempts have been made to conduct otic induction experiments on cell monolayers, rather than on three-dimensional cultures that are prone to higher variability[13,38]. Marcelo Rivolta’s group obtained otic progenitor cells (expressing PAX8, SOX2, FOXG1, PAX2, NESTIN, SIX1 and GATA3) following 10-12-day-culture of hESC monolayers in SFM containing a combination of FGF3 and FGF10 or combinations of EGF, IGF-1 and bFGF factors. The authors described two different types of colonies, large epithelioid colonies, composed of flat cells of large cytoplasm (otic epithelial progenitors, OEPs) and smaller colonies, formed by cells that presented denser chromatin and cytoplasmic projections (otic neural progenitors, ONPs). HC-like cells that co-expressed various HC marker combinations were obtained from OEPs following culture in SFM containing EGF and RA. A similar protocol was applied by Chen et al to generate OEPs and ONPs from hiPSCs. OEPs were grown on mitomycin-treated chicken embryonic utricle stromal cells, in SFM containing EGF and RA, to yield rates of over 40% of cells that co-expressed HC markers (BRN3C, MYOSIN VIIA and ATOH1) and demonstrated some other characteristics of HCs such as the presence of mechano-transduction channels and some electrophysiological activity. In in vitro co-cultures of OEP-derived HC-like cells and SGNs from neonatal mice, SGNs extended neurites to the induced HC-like cells and formed active synapses. Additionally, OEPs were transplanted into the scala tympani of Slc26a4-null mice that present HC defects. At 4 wk post-transplantation some cells had migrated to the scala media and had integrated into the damaged epithelium, expressing MYOSIN VIIA and forming synaptic connections with native SGNs. The same protocol was applied by Azel Zine’s group to obtain HC-like cells from hiPSCs; otic induction was significantly increased when the EGF/RA step was substituted by treatment with a Notch inhibitor, in agreement with accumulated evidence that Notch plays a key role in the differentiation of sensory otic lineages. Thus ATOH1 expression was much higher in cultures exposed to the Notch inhibitor and around 50% of the cells in these cultures expressed MYOSIN VIIA, as opposed to cultures grown in the presence of EGF/RA, where the percentage of cells expressing this HC marker did not reach 5%. Unfortunately, no hair bundle formation was detected on differentiating cells. Very importantly, hiPSC-derived otic progenitors could survive in an in vivo ototoxic damage model. The cells were implanted into the cochlea of adult guinea pigs that had undergone amikacin treatment. Two weeks after implantation, surviving progenitors had engrafted within the damaged cochlear sensory epithelium and expressed MYOSIN VIIA; some expressed SOX2, pointing at their differentiation towards a SC type. Interestingly, those progenitors that had integrated outside the area of the OC did not express MYOSIN VIIA. Similar results were obtained when implanting murine otic progenitors into the same in vivo model.
In addition to the protocols discussed above, other HC induction protocols have been described. An example is provided by the work by Ouji and colleagues[41,42], based on the culture of mESC-derived embryoid bodies in medium conditioned by ST2 stromal cells. This treatment led to the induction of HC marker expression and the formation of stereocilia-like structures in some of the cells; additionally, some cells were shown to integrate into developing chick otocysts. A simpler method was developed by Ohnishi et al in an attempt to eliminate the need for conditioned media, complex growth factor combinations, or the use of xenogeneic cells. They reported expression of MYOSIN VIIA and βIII TUBULIN proteins in hiPSC cultures that had been grown in defined medium, using bFGF as sole growth factor; stereocilia-like protrusions were observed in some MYOSIN VIIA-expressing cells. Although simpler than other methods, induction rates were extremely low.
Differently from the methods described above, Domingos Henrique’s group directly programmed mESCs to become HCs by forcing the simultaneous expression of Gfi1, Pou4f3 and Atoh1 (GPA), coding for three key transcription factors in HC development. Theirs was an extremely fast and efficient induction protocol that in 8-12 days yielded large numbers of cells (54% ± 2%) that co-expressed various HC markers. Addition of RA or inhibition of the Notch pathway during GPA overexpression resulted in increased HC induction rates (84% ± 1% and 70% ± 2%, respectively). Some maturation of the MYOSIN VIIA+ cells were observed from d8 to d12, indicated by a decline in SOX2 expression and clear expression of the hair bundle proteins ESPIN and CADHERIN23 in membrane protrusions that did not reach the degree of organization found in normal HC stereociliary bundles. Nevertheless, FM1-43 incorporation experiments pointed at the presence of potentially functional mechano-transduction channels. Reyes et al also resorted to genetic modification of mESCs in order to guide their differentiation in vitro and attained high rates of differentiation of mESCs to glutamatergic neurons through the transient expression of Ngn1 in the cultures.
ESCs and iPSCs have also been differentiated in vitro[13,45] and in vivo towards SGNs. Some of the work has consisted on generating stem cell-derived NPs that have then been implanted in the inner ear to promote their differentiation towards the SGN lineage. An example of this approach is the work carried out by Corrales et al, who grafted mESC-NPs into the cochlear nerve trunk of ouabain-treated gerbils. Implanted cells survived and demonstrated βIII TUBULIN and PERIPHERIN expression; interestingly, they extended processes towards the denervated HCs in the OC, indicating a role of the host environment as provider of survival, differentiation and guidance cues. Unfortunately, no functional recovery could be demonstrated. Coleman et al implanted mESC-NPs into the scala tympani of chemically deafened guinea pigs, selecting a delivery route that was clinically more relevant than others previously used, such as direct injection into the auditory nerve. Transplanted cells were observed in the scala tympani of transplanted hosts at 4 wk post-transplantation. mESC-derived cells were also observed in Rosenthal’s canal, close to surviving endogenous SGNs, although their numbers were extremely low, indicating that delivery into the scala tympani was not an efficient route to direct exogenous cells to the Rosenthal’s canal. Sekiya et al transplanted mESCs that had been exposed to the neuralizing activity of stromal cell-derived medium into the internal auditory meatal portion of the auditory nerve, aiming at minimizing the risk of damage to the cochlea and optimizing delivery to the target site. The group observed migration of the implanted cells along the damaged auditory nerve, into the Rosenthal´s canal and to the scala media. Interestingly, no significant migration was observed when mESC-NPs were implanted in intact auditory nerves; instead, the cells extended numerous neuritic processes along the nerve. These observations, together with the fact that implanted cells exhibited varying morphologies depending on their location, pointed at an interaction of the exogenous cells with local environmental cues. In another series of experiments, Okano et al recorded higher rates of exogenous cell survival when implanting mESC-NPs in the modiolus of deafened guinea pigs, compared to their implantation in non-injured ears. Surviving cells differentiated to neurons that extended projections towards peripheral and central auditory targets. Interestingly, although synapse formation could not be demonstrated, some functional recovery was observed in some animals.
Work carried out by Albert Edge’s group on hESCs identified BMP4 as a critical molecule to differentiate hESCs towards SGNs. When implanted in an in vivo gerbil model, hESC-derived NPs differentiated and engrafted in the auditory nerve trunk. The neurons extended projections to the sensory cochlear epithelium and towards the brain stem. Unfortunately, synapse formation could not be demonstrated. As mentioned above, hESC-NPs were also generated in Rivolta’s laboratory (otic neural progenitors, ONPs). ONPs were transplanted into the modiolus of ouabain-treated gerbils; implanted cells survived and formed an ectopic ganglion in the modiolus, with neurons that extended neurite projections to the OC. At 10 wk post-implantation some of the hESC-derived neurons had migrated from the ectopic ganglion to the Rosenthal’s canal and some cells were seen migrating towards the brainstem; SYNAPTOPHYSIN staining pointed to the establishment of synaptic connections of hESC-derived neurons with neurons in the cochlear nucleus. Importantly, functional tests carried out at 4 wk post-transplantation demonstrated an improvement in ABR thresholds of animals that had received ONPs; functional restoration correlated to the increase in neural density resulting from ONP transplantation. ONPs were also obtained from hiPSCs and could be differentiated to neurons expressing combinations of sensory neuron and other neuronal markers (βIII-TUBULIN, BRN3A, NF200, NEUROD1, ISLET1). These neurons established active synapses in co-cultures with HC-like cells that were also generated from hiPSC cultures.
Survival of NPs derived from miPSCs has also been demonstrated in vivo, following transplantation into mouse cochleae; some of the surviving cells expressed the glutamatergic neuron marker VGLUT1 and were seen to project neurites towards cochlear HCs. Differently to the approaches described above, Ishikawa et al differentiated hiPSC-NPs to neurons in vitro, prior to their transplantation. Although the cultures contained a mixture of neuronal types and they were at various stages of maturation, around 95% of the cells expressed VGLUT1. The authors conducted parallel differentiation experiments on Matrigel-coated plates and on 3D collagen matrices, obtaining similar results. Implantation of 3D cultures into the scala tympani of normal hearing-competent guinea pigs demonstrated differentiation of hiPSC-NPs to glutamatergic neurons although there was a significant decline in the number of surviving exogenous cells during the first two weeks following transplantation. Based on the loss of Oct3/4 expression in differentiated cultures, the authors defended the safety of their approach, since one of the risks posed by the transplantation of undifferentiated cell types such as ESCs and iPSCs is their potential to give rise to tumours. Nevertheless, the risk of tumour formation by implanted cells cannot be completely eliminated, and thus efforts have also been made to obtain otic sensory cell-like cells from fully differentiated somatic cell types that may overcome this problem. In line with this argument, Durán-Alonso et al applied to cultures of human fibroblasts the direct conversion protocol described by Costa et al. Over-expression of the GPA combination of transcription factors induced the expression of HC markers MYOSIN VIIA, BRN3C and ESPIN. Despite good transduction rates and a strong increase in HC gene transcript expression, clear morphological changes and expression of a combination of HC proteins (MYOSIN VIIA, ANNEXIN A4, ESPIN) was only observed when transduced cells were cultured in SFM containing EGF and RA, as employed by Rivolta’s group on hFASCs[13,28]; however, cell polarization or formation of stereocilia-like protrusions were not observed. Transcriptomic analyses of these cultures indicated an enrichment of genes related to HC development and differentiation, together with genes involved in neuronal differentiation.
Organoids: Some recent work has resulted in the establishment of 3D inner ear organoid cultures[52-56], where differentiation of ESCs and iPSCs is conducted under culture conditions that sequentially recreate the stages leading to the development of various inner ear cell types in vivo. Thus the cultures are exposed to combinations of factors that activate and inhibit key signalling pathways to ultimately render the step-wise formation of non-neural ectoderm, pre-placodal and otic placodal epithelia, and otic vesicle epithelium that ultimately gives rise to HCs, SCs and sensory neurons[52,54,57]. Differentiating cultures are provided with added extracellular matrix proteins that support the self-organisation of the cells into biologically more relevant 3D cultures than those growing as monolayers on tissue culture plates. This arrangement yields clusters of HC-like cells that express an array of HC markers and exhibit basal-to-apical polarization, ESPIN-labelled hair bundles containing functional mechano-transduction channels, and a diversity of voltage-dependent currents. Neurons also emerge within these cultures that establish synaptic contacts with developing HCs[52,57]. Most of the work that has been carried out to date on inner ear organoids has focused on dissecting the identities and the complex interactions of the signalling pathways that regulate inner ear development[57-59], but the numerous advantages offered by these cultures, in terms of cellular complexity, cell phenotype maturation and numbers of induced cells make them ideal substrates for other important applications in the field[60,61]. At present there are important shortcomings to the use of inner ear organoids as sources for HC transplantation into the cochlea. A major hurdle is the fact that the applied protocols yield vestibular HC types[56,62]. Work is underway to elucidate what elements are missing in the current cultures that may yield cochlear HCs; Jeong et al have recently described a series of modifications to the original method that result in the generation of various HC types in organoid cultures, some expressing cochlear HC markers. On the other hand, inner ear organoids may already constitute valid substrates to investigate SGN development and initiate studies towards a possible application in approaches to SGN regeneration. Perny et al have modified initially published protocols, obtaining mESC-derived cultures that contain a large number of neurons that express a whole array of sensory otic neuron markers (GATA3, PROX1, ISLET1, p75, MAFB, PERIPHERIN) and display electrophysiological properties similar to those of SGNs. Very interestingly, characterization of the neurons present in these cultures indicates that these are not vestibular neurons but cochlear SGNs.
Summarizing what has been discussed above, a number of exogenous cell types have been evaluated for their potential to replace damaged cells in the adult mammalian cochlea, in a quest for a promising approach to hearing restoration. Some of these results are summarized in Figure 2.
Figure 2 Overview of exogenous donor cell types that have been tested for their potential to give rise to hair cell- and / or spiral ganglion neuron-like cells.
Advantages and disadvantages of the various donor cell types are indicated, as well as the main results that have been obtained with these cells in in vivo and in vitro studies. HC: Hair cell; SGNs: Spiral ganglion neurons.