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First published online July 26, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2685 -2694
doi:10.1634/stemcells.2007-0393; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Concise Review: The Potential of Stem Cells for Auditory Neuron Generation and Replacement

Bryony Colemana,b,c, Michelle G. de Silvad,e, Robert K. Shepherda,c

aThe Department of Otolaryngology, University of Melbourne, East Melbourne, Victoria, Australia;
bThe Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia;
cThe Bionic Ear Institute, East Melbourne, Victoria, Australia;
dDepartment of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria, Australia;
eMurdoch Childrens Research Institute, The University of Melbourne, Parkville, Victoria, Australia

Key Words. Stem cells • Deafness • Differentiation • Auditory neuron • Cell transplantation • Cochlear implant

Correspondence: Bryony Coleman, Ph.D., Department of Otolaryngology, Royal Victorian Eye and Ear Hospital, Level 2, 32 Gisborne Street, East Melbourne 3002, Victoria, Australia. Telephone: +61 3 9929 8384; Fax: +61 3 9663 1958; e-mail: bcoleman{at}bionicear.org

Received on May 22, 2007; accepted for publication on July 19, 2007.

First published online in STEM CELLS EXPRESS  July 26, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
Sensory hair cells in the mammalian cochlea are sensitive to many insults including loud noise, ototoxic drugs, and ageing. Damage to these hair cells results in deafness and sets in place a number of irreversible changes that eventually result in the progressive degeneration of auditory neurons, the target cells of the cochlear implant. Techniques designed to preserve the density and integrity of auditory neurons in the deafened cochlea are envisaged to provide improved outcomes for cochlear implant recipients. This review examines the potential of embryonic stem cells to generate new neurons for the deafened mammalian cochlea, including the directed differentiation of stem cells toward a sensory neural lineage and the engraftment of exogenous stem cells into the deafened auditory system. Although still in its infancy the aim of this therapy is to restore a critical number of auditory neurons, thereby improving the benefits derived from a cochlear implant.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
Cochlear implants are designed to electrically stimulate the auditory nerve in severe to profoundly deaf individuals. These neural prostheses bypass the nonfunctional component of the damaged auditory system, the hair cells, and directly depolarize auditory neurons to effectively restore the sound transduction pathway. Technological advances and improved speech processing strategies have resulted in better clinical performance with these devices over time. However, there is a high degree of variation in individual patient outcomes, with some cochlear implant recipients achieving significant improvements in auditory performance, whereas others experience minimal benefits [1–3]. Several factors are likely to contribute to this variation, including etiology, duration of deafness, and age of implantation [4]. There is also a positive correlation between auditory performance and duration of implant, indicating that performance improves with experience with this device [4]. One factor that remains controversial is whether greater numbers of auditory neurons can improve clinical performance with a cochlear implant, given that these neurons undergo progressive degeneration following deafness. Although animal studies indicate that ongoing degeneration of the auditory nerve may compromise cochlear implant function [5, 6], the limited studies directly comparing clinical performance with the number of surviving auditory neurons report no correlation [7–9]. These conflicting data suggest that clinical performance indicators remain to be elucidated, and it is likely that the density and integrity of auditory neurons along the length of the cochlea (rather than absolute number) are likely to effect the performance of this neural prosthesis [10].

Several research strategies are currently directed toward cell transplants to restore or replace the degenerating neural elements following hearing loss. Stem cells are excellent candidates for this therapy, as they have the potential to increase the number of auditory neurons available for electrical excitation via a cochlear implant. The success of such a therapy will critically require the conversion of embryonic stem cells to neurectoderm progenitors, the directed differentiation of these progenitors into auditory neurons, the delivery of cells into their target site within the delicate cochlea, and the successful integration of transplanted cells with existing neural populations. To date, such work has been limited to experimental studies on animals, and the majority of these studies uses stem cells of murine origin. The following discussion is therefore directed at reviewing the strategies used for directed differentiation of mouse embryonic stem cells toward a neural fate, conversion into sensory neural precursors, and transplantation of stem cells into the deafened mammalian cochlea. To facilitate the review for nonauditory scientists, the basic anatomical structures of the inner ear are illustrated in Figure 1.


Figure 1
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  		Figure 1. Anatomy of the inner ear and cochlea. Schematic illustrations show the anatomy of the mammalian cochlea in transverse section. (A): The cochlea is embedded in the temporal bone of the skull. The auditory nerve is located in the center of the cochlea and is comprised of auditory neurons. The cell bodies of these auditory neurons reside in discrete compartments termed Rosenthal's canal (black shaded areas). The auditory nerve and Rosenthal's canal compartments are collectively referred to as the modiolus. Encircling the modiolus are three fluid-filled chambers termed scala vestibuli, scala media, and scala tympani. (B): The boxed area in (A) is shown in more detail and illustrates the location of auditory neurons within Rosenthal's canal. Each auditory neuron extends one process centrally (to auditory centers in the brain) and one process peripherally (to the sensory epithelium). The sensory epithelium comprises four rows of hair cells that are attached via stereocilia to the tectorial membrane. These hair cells and their supporting cells are collectively termed the organ of Corti (circled). In normal-hearing individuals, the inner hair cells transduce sound waves into electrical signals that are carried to the brain via the auditory neurons. (C): Hearing loss occurs following irreversible damage to the hair cells and degeneration of the organ of Corti (circled). The loss of hair cells causes secondary degeneration of auditory neurons, beginning with a loss of the peripheral processes of these neurons (perforated lines). Abbreviations: AN, auditory nerve; ANs, auditory neurons; RC, Rosenthal's canal; SM, scala media; ST, scala tympani; SV, scala vestibuli.

 

   DIRECTED DIFFERENTIATION OF EMBRYONIC STEM CELLS TOWARD A NEURAL LINEAGE
Top
 Abstract
 Introduction
 Directed Differentiation of...
Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cells are isolated from the inner cell mass of early embryos and possess three general properties that make them excellent candidates for cell replacement therapies. They are capable of self-renewal over long periods of time through continuous cell division, they remain in an undifferentiated state, and they can be induced to form more specialized cell types under controlled physiological and experimental conditions. In order to recapitulate in vivo neural differentiation, embryonic stem cells must initially be induced to form ectoderm tissue. This is initiated by removal of leukemia inhibitory factor from the medium and suspension culture to promote the formation of embryoid bodies [11]. Embryoid bodies are typically large, spherical, floating aggregates formed from dissociated embryonic stem cells. The gene expression patterns in these embryoid bodies are similar to those reported during embryo development [12, 13], and embryoid bodies comprise cells from all three germ layers: endoderm, mesoderm, and ectoderm [14]. The process of differentiation within the embryoid body begins with the formation of an outer layer of endoderm-like cells, which is followed by the development of an ectodermal "rim" and mesoderm specification [15]. Although there are several similarities between the gene and protein expression patterns in embryoid bodies and the early processes of in vivo development, the nature of differentiation within embryoid bodies lacks resemblance to the precise timing of differentiation [16] and the intrinsic anterior-posterior and dorsal-ventral organization observed in the developing embryo [17]. Following embryoid body formation, there are several common methods for the directed differentiation of stem cells toward a neural lineage. The following discussion is directed at reviewing these differentiation models in mouse embryonic stem cells and is summarized in Figure 2.


Figure 2
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  		Figure 2. Directed differentiation models for the generation of neurons from MESCs in vitro. This figure summarizes some of the similarities and differences among directed differentiation models to produce neurons from MESCs in vitro. Several techniques have been used to direct the differentiation of MESCs into neuron-like cells, neural precursors, or neurons. Although many of these studies use suspension culture techniques to produce embryoid bodies from which to further differentiate neurons or neural precursors, this can be avoided using genetic manipulation techniques or growth in chemically defined media. Abbreviations: LIF, leukemia inhibitory factor; MESCs, mouse embryonic stem cells.

 
Retinoic Acid Treatment
A well-established method for the generation of neurons from embryonic stem cells is via timed exposure to retinoic acid [18–20]. The molecular mechanism behind retinoic acid-regulated gene expression during development occurs via an intracellular signaling cascade [21]. Briefly, retinoic acid binds to cellular retinoic acid binding protein and is transported to the nucleus, where alternative isoforms of the protein react with specific nuclear retinoic acid receptors. These receptors combine to form either homo- or heterodimers, which in turn bind to specific sites on the DNA binding domain and result in transcription of the target gene.

Importantly, the treatment of embryonic stem cells with retinoic acid has been reported to promote the expression of neural genes while repressing mesodermal genes [22]. Similarly, overexpression of Stra13 (a retinoic acid target gene) promotes neural differentiation while inhibiting mesodermal differentiation in embryonal carcinoma cells [23]. These findings suggest that retinoic acid treatment may be central to directing the early stages of differentiation toward a neuronal lineage. The upregulation of retinoic acid receptor mRNAs during aggregation-mediated neurectoderm differentiation (concurrent with no detectable changes in endodermal or mesodermal marker mRNA expression levels) implies an important role for retinoic acid during early neuronal differentiation [24, 25].

In vitro application of retinoic acid to 4-day-old embryoid bodies has resulted in improved induction of neuronal differentiation [18–20]. Notably, the rate of spontaneous neuronal differentiation in embryoid bodies was reported to be between 15% and 30%, and this rate was significantly increased to almost 100% following retinoic acid application [20]. In addition, retinoic acid-induced differentiation into neuronal cells was characterized by specific immunocytochemical markers and gene expression that paralleled that of postmitotic neurons [18, 20]. Activation of neuron-specific genes occurred concomitant to expression of calcium, sodium, and potassium ion channels, and differentiated neurons were capable of generating action potentials [18, 20, 26, 27]. Moreover, cultured mouse embryonic stem cells treated with retinoic acid were also observed to form synapses and develop neuronal polarity in vitro [28]. Collectively, these experiments illustrate that functional neurons can be generated from embryonic stem cells in vitro via retinoic acid treatment.

Preconditioned Media
In 1999, Rathjen and colleagues [29] identified a preconditioned medium (MedII, collected from cultured hepatocellular carcinoma [HepG2] cells [30]), which caused the conversion of embryoid bodies to a morphologically distinct cell population that they termed early primitive ectoderm-like (EPL) cells. These EPL cells were found to be similar in morphology, gene expression, cytokine responsiveness, and differentiation potential to primitive ectoderm in vivo. Early primitive ectoderm-like embryoid bodies were irregularly shaped and showed advanced cell differentiation in comparison with embryonic stem cell embryoid bodies [31]. Moreover, EPL cells were observed to differentiate into primitive ectoderm (neurectoderm, neural plate, and neural tube), with almost complete inhibition of endoderm and mesoderm differentiation [32]. The production of a homogenous population of neural precursors in vitro represented an important advance in the directed differentiation of embryonic stem cells into defined neural lineages, in particular for the generation of implantable cells for therapeutic use.

Recently, mouse embryonic stem cells were directed to differentiate into neurons using preconditioned medium from astrocytes [33]. In this study, the authors report the rapid generation of dopaminergic neurons using a 9-day induction protocol, without the production of either astrocytes or oligodendrocytes. These data illustrate the potential of soluble factors in preconditioned media for directed differentiation protocols.

Coculture
A similar method uses the coculture of stem cells with another cell type (or whole tissues) in order to provide the cohort of biochemical cues required for directed differentiation of stem cells into a specific cell type. In vitro, coculture models provide an ideal starting point for differentiation studies in systems where little is known about the precise combination of factors that induce differentiation into a specific cell type. The interactions and signaling between developing tissues play an essential role in regulating differentiation in vivo. Given the complexity of cellular signaling pathways in vivo, this environment is often difficult to mimic completely in vitro. Coculture models offer a method to study differentiation under controlled conditions in vitro with the advantage of being able to replicate some tissue-derived signaling. Coculture models have been used successfully to direct the differentiation of stem or precursor cells into neurons [34–37], hematopoietic cells [38, 39], photoreceptor cells [40], and hepatocytes [41] and to promote cell survival and expansion in vitro [42–45]. Notably, the first published attempt to direct the differentiation of mouse embryonic stem cells toward an auditory neuron lineage used coculture with early postnatal cochlear tissues to improve the number of bipolar neurons generated [37].

Growth Factors and Chemically Defined Media
Neurotrophic factors are able to promote the survival and differentiation of embryonic stem cells into neurons in vitro [46]. Several studies have now demonstrated that differentiation of mouse embryonic stem cells into neurons can be directed by temporal exposure to growth factors and/or neurotrophins in a defined medium [27, 46–51]. Such studies have resulted in the production of neural precursors [48, 49], functional postmitotic neurons [27, 47], and dopaminergic neurons [46, 51] following the removal of serum and treatment with various neurotrophic agents, including basic fibroblast growth factor [27, 48, 49, 51], epidermal growth factor [48], transforming growth factor [46], and glial cell-derived neurotrophic factor [46, 50]. Although these studies differ in the combination and timing of growth factor treatment, all make use of an initial aggregation step (embryoid body formation) prior to treatment, a protocol originally believed to be necessary for the production of neurons. Interestingly, several studies have now illustrated that embryonic stem cells can give rise to neurectodermal precursors or neurons without embryoid body formation by chemically defined low-density culture conditions [52], stromal cell-derived inducing activity [53, 54], or adherent monoculture [55]. Such studies support the use of chemically defined media for cultivation of specific cell lineages when specific inductive factors are known.

Genetic Manipulation
A recent method by which to generate a particular lineage of cells is to "force" differentiation by driving the expression of a gene that is essential to the development of the cell type of interest. Genetic manipulation therefore requires some detailed knowledge of the sequence and timing of gene expression in that cell type. This technique has been elegantly used to produce dopaminergic neurons, which were generated from the forced expression of the transcription factor Nurr1 [56]. Importantly, these neurons were reported to function in animal models of Parkinson disease [56]. Using similar techniques, Ying and colleagues used a green fluorescent protein knockin at the Sox1 locus in a population of embryonic stem cells [55]. The authors used retinoic acid to initiate differentiation of these genetically modified cells, which resulted in the production of a mixed population of neural progenitors. The neural progenitors that expressed Sox1 also expressed green fluorescent protein, thereby enabling the purification of the neuronal population of cells via fluorescence-activated cell sorting. The primary advantage of genetic manipulation techniques is that they are reported to generate a highly purified population of cells, a method that will be essential for future transplantation studies and that is difficult to produce using other neural differentiation protocols.

Combined Differentiation Models
Although each differentiation protocol has individual advantages, it seems likely that future directed differentiation studies will incorporate several of these models in order to more successfully produce homogenous populations of cells for transplantation. An example of such a combined induction method was recently described for the production of dopaminergic neurons from mouse embryonic stem cells [57]. In this study, the authors combined genetic manipulation, coculture, and growth factor treatment to produce a population of dopaminergic neurons for the treatment of Parkinson disease. The neurons generated from this combined differentiation regimen displayed several of the characteristic properties of dopaminergic neurons in vivo, including the expression of tyrosine hydroxylase (~90%) and dopamine, and the electrophysiological properties of midbrain dopaminergic neurons [57]. In addition, these neurons were reported to functionally integrate into the striatum following their implantation in mice. In future, the combination of genetic manipulation with in vitro differentiation techniques may improve the production of homogeneous cell populations suitable for transplantation.


   DIFFERENTIATION OF EMBRYONIC STEM CELLS TOWARD A SENSORY NEURAL LINEAGE
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
In higher vertebrates, all sensory neurons are derived from precursors originating in the placodes of the head or from the neural crest. Importantly, the differentiation of sensory neurons from both the placodes and the neural crest is likely to be similar, given that the same transcription factors and regulation appear to be used in both regions [58]. For example, neurogenin 1 and 2 are expressed in precursors of both placode- and neural crest-derived sensory neurons [59, 60]. Considering that the neural crest also gives rise to autonomic neurons, mechanisms must exist to induce this sensory (rather than autonomic) fate [58]. Notably, neurogenin 1 and 2 are not expressed in autonomic precursors that derive from the neural crest [61], and targeted deletion experiments have illustrated that neurogenin 1 and 2 expression is critical for the development of sensory, but not autonomic, ganglia [58, 62, 63]. Furthermore, several loss-of-function experiments have illustrated the importance of the proneural genes neurogenin 1 [64] and NeuroD [65, 66] in normal auditory development. Notably, although NeuroD expression is sufficient to drive survival and differentiation of sensory neural precursors, it remains unclear what drives the initial expression of its upstream regulator neurogenin 1.

For stem cell therapy in the deaf cochlea to be effective, a more thorough understanding of the factors causing differentiation into auditory neurons is required. Although this is an emerging area of research, a recent publication describes the generation of greater numbers of stem cell-derived neurons following retinoic acid treatment and coculture [37]. More specifically, the authors report that conditioned medium from postnatal cochlear tissues significantly improved the number of bipolar neurons in comparison with untreated controls. These promising findings suggest that tissue-derived signaling may provide key information in the directed differentiation of embryonic stem cells toward an auditory neural lineage. The elucidation of these soluble factors will likely aid in the generation of greater numbers of auditory neurons from stem cells in vitro. The sequential purification and precise characterization of these neurons in vitro will be essential for determining their full potential for in vivo transplantation.


   CLINICAL CONSIDERATIONS FOR THE TRANSPLANTATION OF STEM CELLS FOR SENSORINEURAL HEARING LOSS
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
Although a small number of stem cell therapies have been investigated for hair cell replacement [67–70], the majority of stem cell transplantation studies have been directed at the replacement of degenerating auditory neurons [71–80]. The major findings from these studies are summarized in Table 1Go, and the reader is referred back to Figure 1 for anatomical details.


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  		Table 1. Summary of stem cell transplantation studies for auditory neuron generation and replacement

 


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  		Table 1. (continued)

 
Several stem cell types have now been delivered into the mammalian cochlea for the replacement of auditory neurons, including bone marrow stem cells [72, 81], neural stem cells [73, 74, 76, 77], and embryonic stem cells [71, 74, 75, 78–80]. Although transplantation methodology varies among publications, all authors report the survival of exogenous stem cells in the inner ear for periods of between 3 and 13 weeks. In addition, several of these studies report the dispersal of transplanted cells throughout the cochlea [72, 75, 76, 78, 79, 82] and limited detection within the target site, Rosenthal's canal [78, 79, 83, 84]. Notably, a recent publication described several advances in stem cell delivery into the cochlea, including the extended survival of transplanted cells and the extensive migration along the auditory nerve [80]. Although such findings illustrate the promise of this therapy, many questions remain unanswered, including the stage of differentiation at which these cells should be transplanted, the site of their transplantation and the development of safe surgical techniques that can be applied clinically, the successful incorporation of exogenous cells into the host without being rejected, and the functional connectivity of transplanted cells, including their ability to form new synapses with existing neurons in an appropriate cochleotopic manner.

Cellular Heterogeneity: To Differentiate or Not To Differentiate?
Despite the differentiation potential of embryonic stem cells, initial attempts to obtain specific cell types using differentiation protocols have been slow due to limited knowledge of the growth factor combinations and developmental control genes involved in lineage restricted differentiation [16]. Furthermore, the differentiation of a homogeneous population of cells has been problematic due to the routine production of multiple cell types using differentiation protocols [85]. For cell transplantation studies to be successful, it will be important to establish robust protocols for the production of pure populations of cells [16, 47, 85, 86].

A major challenge of embryonic stem cell research is the stage of differentiation at which cells should be delivered in vivo. Although there are suggestions that embryonic stem cells will incorporate into their host environment with limited ex vivo differentiation [87, 88], this method has a considerable drawback: undifferentiated stem cells possess similar properties to tumors and, when transplanted into animal models, have been reported to form teratocarcinomas [89, 90]. According to Sell [91], the answer to avoiding tumor formation in vivo is to differentiate cells prior to transplantation. The predifferentiation of stem cells appears to greatly reduce the formation of teratocarcinomas in vivo, as is illustrated in several animal studies [56, 92–94]. However, one study reported tumor formation at the site of implantation regardless of predifferentiation [95]; these authors suggested that the host environment played a critical role in tumor formation. In addition, relying on the local environment to produce the cues necessary for directed differentiation of the cell of choice poses risks in itself, as the degree of pathology is likely to vary from one individual to the next, and, as such, exert a different effect on the differentiation of transplanted cells. Notably, several in vivo studies that predifferentiate embryonic stem cells into neurons prior to transplantation have reported functional incorporation of those neurons into the injured central nervous system [56, 96–98].

More recently, MacLaren and colleagues compared the ability of retinal precursor cells taken from embryonic, postnatal, and adult animals to form functional connections following transplantation into the degenerating retina [99]. Notably, the authors report that cells derived from postnatal retinas survived in vivo, showed robust integration, differentiated, formed synaptic connections, and improved visual function following transplantation [99]. Conversely, the cells derived from embryonic retinas (consisting almost entirely of proliferating progenitors) failed to integrate, as did cells taken from adult retinas, even though both cell types survived for a maximum period of 3 weeks in vivo. These results convey important new information to studies investigating the replacement of neurons using stem cells. Specifically, this work suggests that successful nerve replacement is likely to require cells at a specific stage of differentiation and that simply engrafting precursor cells into the lesion site may not promote recovery of function. Although less differentiated cells are preferable for avoiding immunorejection, more differentiated cells may possess the capacity to transfer information correctly to the brain. The function of newly transplanted cells is paramount to the success of cell transplantation in the nervous system, and this is discussed below in Synaptogenesis and Cochleotopic Connectivity.

Delivery of Stem Cells to Their Target Site
A further challenge lies in the surgical delivery of stem cells into the deaf cochlea. The primary goal of such surgery is to achieve an even distribution of neurons along the length of the cochlea while minimizing damage to the delicate cochlear architecture. Although there are now numerous published accounts describing stem cell transplantation into the mammalian cochlea, generally it can be divided into two categories based upon the surgical delivery site: either into the fluid-filled compartments of the cochlea [75, 76, 79, 83, 84, 100] or directly into the modiolus or auditory nerve [71–74, 77, 78, 101]. Although the latter technique allows for the more targeted delivery of cells, it can result in mechanical damage to the cochlea [71, 101], thereby causing further degeneration of residual auditory neurons. In comparison, the former technique causes minimal trauma to the cochlea; however, cells delivered in this manner have been shown to disperse throughout the cochlea [76, 79, 82] with only small numbers reaching the target site of Rosenthal's canal [75, 79]. The most recent publication describing the delivery of stem cells into the auditory nerve [80] reflects several advances in relation to previous studies. Specifically, the authors describe the survival of transplanted cells for at least 13 weeks in vivo (the longest to date) and the extensive migration of these cells from the base of the auditory nerve (the injection site) toward the apex. Furthermore, the transplanted stem cells expressed neural marker β III tubulin, were contained within the auditory nerve, and extended processes toward their peripheral targets (hair cells). Such findings are indeed promising for auditory neuron replacement using stem cells, particularly when considering the combined application of electrical stimulation via a cochlear implant (inserted adjacent to the auditory nerve). In this way, electrical stimulation from a cochlear implant may serve to activate both the residual and new population of auditory neurons, resulting in improved speech perception and language outcomes for severely to profoundly deaf individuals.

Immunocompatibility of Stem Cells
It is likely that differentiating stem cell populations will express human leukocyte antigens (HLA) and may therefore be rejected after transplantation [87]. Despite this suggestion, evidence from early studies on human embryos suggests that embryonic stem cells may be "immune privileged," as they do not express HLA proteins [102, 103]. A more recent study supports these suggestions, finding only a moderate increase in HLA expression following differentiation of human embryonic stem cells [104]. This group recently demonstrated that transplanted human embryonic stem cells (undifferentiated and differentiated) display a very low susceptibility to immunorejection, a characteristic that might make them good candidates for transplantation due to their reduced requirement for the administration of immunosuppressive drugs [105]. Although immunosuppressive drugs could be used to inhibit rejection of transplanted cells, this is still far from ideal, as these treatments can result in several complications including impaired wound healing, opportunistic infections, drug-related toxicities, skin malignancies, and low-grade lymphomas [106].

One solution to this problem may be to genetically engineer stem cells without major histocompatibility complex (MHC) proteins to decrease their immunogenicity. When performed in mouse embryonic stem cells, however, skin grafts that were deficient in both MHC class I and class II antigens were still rejected [107]. It may therefore be necessary to "knock out" and then "knock in" the desired MHC genes so that grafts are not rejected by the host [108]. Advances in nuclear transfer have now made it possible to "customize" stem cells for individuals. Somatic cell nuclear transfer uses nuclear transfer techniques to remove the nucleus from an adult donor cell and introduce it into an enucleated donor oocyte, thereby producing a population of pluripotent stem cells with the genome of the donor nucleus [109]. Cells produced from this technique can then be propagated in large numbers for transplantation. Importantly, cells generated via nuclear transfer are autologous and therefore avoid immunorejection and the requirement for immunosuppressive medication [15].

Synaptogenesis and Cochleotopic Connectivity
For stem cells to form functional connections with second order neurons in the cochlear nucleus, they must be capable of forming new synapses, the structures permitting signaling between nerve cells. Synaptogenesis is characterized by three key events: the formation of selective connections between a developing axon and its target cell, the differentiation of the developing axon's growth cone into a presynaptic terminal, and the development of the appropriate postsynaptic apparatus in the target cell [110]. The coordination of synaptogenesis is believed to be critically dependent on the intercellular signaling interactions between the developing axon and the target, including the recognition of an appropriate postsynaptic cell and the sequential differentiation of pre- and postsynaptic elements [110]. Indeed, the selection of synaptic partners is a crucial step in the formation of a functional neural circuit [111–113]. Interestingly, recent evidence indicates that transcription factors might be a central feature in nervous system wiring [114]. Specifically, the authors consider that transcription factors may specify connectivity by regulating the expression of genes that determine the responsiveness of the neuron to axon guidance and synaptic molecules. An improved understanding of the transcription factors involved in auditory system development may therefore help to direct stem cell-derived auditory neurons to their appropriate targets in situ.

In the context of auditory neuron replacement using exogenous neurons, the regeneration of auditory nerve fibers and the formation of new synapses will need to occur in a cochleotopic fashion with endogenous neurons in the cochlear nucleus. The residual population of auditory neurons that persist even after prolonged periods of deafness may therefore be critical in providing cues to direct the growth of these new axons; however, this remains to be illustrated experimentally. Given the infancy of this niche of research, there are currently no published accounts of attempts to direct the growth of newly transplanted neurons toward central (or peripheral) targets in the cochlea. This is likely to be the critical step in the development of a useful cell-replacement therapy, particularly considering that synaptic patterning in the vertebrate nervous system is essentially influenced by synaptic activity [115, 116]. In this respect, electrical stimulation via a cochlear implant may play a fundamental role in the reconnection of newly transplanted neurons by providing the stimulus they require for functionality.


   DEVELOPING CELL TRANSPLANTATION STRATEGIES FOR CENTRAL NERVOUS SYSTEM INJURY AND DISEASE
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 
There are now numerous published accounts describing the delivery of mouse embryonic stem cell-derived neurons and neural precursors (generated ex vivo) into the central nervous system [56, 93, 96–98]. Moreover, several of these studies have reported integration [56, 96–98] and partial recovery of function in animal models of disease [56, 93]. Although these studies are promising for future tissue replacement and repair in vivo, recent clinical studies have highlighted that there are still several problems that need thorough investigation before patient outcomes can be improved. Specifically, in clinical trials for Parkinson disease, improvements have been highly variable, with some patients showing an excellent response (40%–60% improvement in motor function scores) and others showing little or no improvement [117]. The survival and growth of grafted neurons are cited as being important determinants in outcome variability [117, 118], and future therapies are likely to involve the coadministration of trophic support and/or growth factors in an attempt to improve the survival of newly transplanted neurons and surrounding cells in the damaged area [118]. In addition, there is a requirement to obtain a high yield and pure population of the cell type required for transplantation [119]. According to these authors, nonpurified neural derivatives pose a danger to clinical transplantation studies, as there are currently no data to indicate whether undifferentiated cells persist in the brain, exit the cell cycle, or divide autonomously, leading to tumor formation [119]. Ultimately, the long-term persistence and function of cell transplants in situ will be an important factor in defining the success of cell transplantation therapies for humans in the future. It is imperative that effective therapies are pursued in concert with developing a thorough understanding of the neurobiology that underlies the functional integration of grafted cells into the damaged nervous system [120] so as to ensure the best possible outcomes for patients.


   OUTLOOK
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
Disclosure of Potential...
 Acknowledgments
 References
 
The successful engraftment of stem cells into the deafened cochlea is a developing and challenging area of auditory neuroscience, which is likely to produce several benefits for cochlear implant recipients in addition to informing related cell replacement therapies in both the peripheral and central nervous systems. However, there are several considerations that require thorough investigation before this therapy becomes clinically feasible.

Specifically, we speculate that embryonic stem cells are likely to be the best candidates for a cell replacement strategy in this system. The elucidation of the precise factors causing auditory neuron differentiation and the timed application of these factors mean that embryonic stem cells could be partially differentiated toward an auditory neuron lineage, delivered into the cochlea, and then the final factors applied to promote terminal differentiation. Such an approach may also facilitate the directed growth of new processes toward central and peripheral targets, a critical step if stem cell-based therapy is to be beneficial. Although adult stem cells are appealing from the perspective of autologous transplantation, new techniques in cell manipulation are likely to mean that patient-specific cells can be created either by masking the human leukocyte antigens or removing them altogether. Moreover, the development of targeted transplantation techniques will be paramount in bringing this therapy to the clinic. These techniques should include the direct delivery of replacement cells into the modiolus while ensuring minimal damage to the auditory nerve and delicate cochlea architecture.

If repeatable protocols for the directed differentiation of human embryonic stem cells into auditory neurons can be established, concomitant to advanced surgical techniques for the delivery of these cells into their target site in the cochlea, then a final challenge will be to direct the growth of these new neurons toward their central and peripheral targets. If this stage can be reached in the laboratory and routinely repeated in multiple vertebrate models of deafness, then we will be close to taking this regenerative therapy into the clinical setting.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
References
 
We acknowledge the financial support of the National Institutes of Health (Contract NIH-N01-DC-3-1005), Department of Otolaryngology at the University of Melbourne, the Bionic Ear Institute, and the ARC Special Research Centres (Particulate Fluids Processing Centre). B.C. is supported by a Wagstaff Fellowship in Otolaryngology from the Royal Victorian Eye and Ear Hospital. Figure 1 was electronically prepared by Andrew Bonollo at the Biomedical Multimedia Unit, University of Melbourne.


   REFERENCES
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 Abstract
 Introduction
 Directed Differentiation of...
 Differentiation of Embryonic...
 Clinical Considerations for the...
 Developing Cell Transplantation...
 Outlook
 Disclosure of Potential...
 Acknowledgments
 References
 

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