When biopsies obtained from patient cells are able to expand and differentiate in culture and after transplantation into the same patient to heal a defect, autologous therapies are feasible. A direct biopsy is taken from tissue that is the same as the engineered tissue type. An indirect biopsy may be taken from a similar tissue when a direct biopsy would cause considerable injury. Autologous transplantation holds the great advantage of avoiding the risk of immune rejection due to differences in histocompatibility antigens. Therefore, immunosuppression is not required. However, there is a substantial practical appeal to “off the shelf” products that do not require the cost and time associated with customized preparation. Autologous transplantation has already achieved regulatory approval and reached the market for patients with life-threatening burns and for large articular cartilage lesions. These successful applications highlight the ongoing efforts to identify new autologous cell-based therapies. The updated opinion states that in addition to traditional bone marrow derived cells, ependymal cells, induced pluripotent stem (iPS) cells, and olfactory sheath cells have been recognized as promising sources. These sources will spur ongoing efforts to evaluate the new cell sources for cell replacement therapy.
A practical alternative to the unavoidable immune rejection is the introduction of autologous cells. Current viable cell sources for autologous inner ear sensory cell replacement are: ependymal cells, iPS cells, olfactory sheath cells, bone marrow cells and stem cells from amniotic fluid and placenta. These cells could be obtained from the recipient themselves via biopsy, as self-made cells share identical major histocompatibility complex with the recipient. This would enable autologous transplantation without immunosuppression.
The immune rejection may be overcome by isolation of donor cells for reintroduction into the same host. Autologous transplantation is a widely accepted and approved technique for regenerative medicine. It has an enviable profile of assets, including lack of immune rejection and is free of disease transmission from donor to recipient. Self-made cells also share identical major histocompatibility complex with the recipient, enabling autologous transplantation without immunosuppression. Exploration of these approaches may serve as preparation towards clinical trials in the near future.
One of the major disadvantages of autologous transplantation is the limitation in the amount of available tissue. Various researchers are striving toward hearing restoration with various substitute cell sources possessing featured properties, not necessarily a replica of the native inner ear sensory cells.
2.1 Ependymal cells
A recent study [40
] reported extensive analyses using structural, molecular, and functional criteria to demonstrate that adult brain germinal zone cells, derived from the same neural ectodermal layer as the otic vesicle epithelial cells, demonstrate intriguing putative hair cells properties and may serve as a novel source of autologous cells for hair cell replacement. In the adult brain of both rodents and human, ependymal cells form a multi-ciliated single cell layer lining the lateral ventricles. These cells share many essential molecular, morphological, and physiological characteristics with inner ear HCs. Further they are polarized with actin-based stereocilia and microtubule-based kinocilia, similar to vestibular hair cells of the inner ear. They can be identified by and commonly used HC markers such as: myosin VIIa (), myosin VI, and CtBP2/RIBEYE. In a fashion similar to hair cells, ependymal cells rapidly uptook the styryl dye FM1–43 that permeates hair cell transduction channels, suggesting that the ependymal cells express similar large conductance, nonselective ion channels. Also notably important, these cells can incorporate into the cochlear sensory epithelia and have several defining electrophysiological characteristics of HCs. Moreover, they are electrically active, send synaptic input to target-deprived SGNs and are capable of releasing glutamate in response to membrane depolarization (). A distinct advantage is that, unlike hair cells, ependymal cells appear to retain the ability to proliferate and proliferative myosin VIIa positive ependymal cells were identified both in vitro
and in vivo
. This raises their prospects for use in autologous replacement of nonrenewable HCs. Further examination of the differences between the proliferative capacity of ependymal cells and the quiescence of hair cells may make ependymal cells a valuable comparative model for understanding some basic biological questions.
Ependymal cells demonstrate characteristics of HCs
Functional connections established between ependymal cells and SGNs
Based on proof of principle experiments established with the functional test of ependymal cells, we assert that ependymal cells can undergo functional switches to assume functional characteristics of HCs. Thus, a broader view of the functional adaptation, or an inborn functional plasticity was just recognized. Some specialized cells may directly adapt their functions upon switching to a new environment, which may act as an alternative to the more complicated cell fate switch. Although the richness and complexity of this functional plasticity remains to be appreciated, an updated concept of essential multipotency of lineage phenotypes should be broadened to include the emerging recognition of multi-functional potential of stem cells.
Application for human therapy depends on further evaluation of their performance in animal models. The potential hurdle for human therapy is the age- and disease-related changes of ependymal cells and safety concerns about collecting these cells. Nonetheless, as a tool for understanding the basic science of cell replacement in the inner ear, this work represents an important step forward [41
]. Understanding of the mechanisms of functional plasticity may open a new window for cell replacement therapy.
2.2 Induced pluripotent Stem (iPS) cells
A successful allograft is accompanied by the insurmountable pitfalls of lifetime immunosuppression. An attractive solution to solve both the shortage of reproducible cells and immune rejection is the autologous source of stem cells, which are collected from the same patient. This will eliminate complications associated with immune rejection, and thus the deleterious side effects of immunosuppressive medications can be avoided. Limitations of harvesting the marketable autologous stem cells make this approach beyond the reach of clinical practice.
Recent ground breaking research has found that activation of a set of stem cell genes can reprogram terminally differentiated cells to acquire properties similar to those of embryonic stem (ES) cells [42
]. The reprogrammed cells are namely induced pluripotent stem (iPS) cells. The iPS cells were similar to ES cells in morphology, proliferation, expression of some ES cell marker genes, as well as the multiple differentiations. This elegant proof of principle experiment shattered the established dogma, that terminally differentiated somatic cells could not be reprogrammed to the embryonic state.
Besides the general characteristics of ES cells, the unique features of iPS cells grant them additional advantages for cell replacement therapy. First, iPS cells could be generated from the customer patient, immune rejection is essentially overcome. Secondly,the application of somatic cell derived iPS cells are free of ethical concerns regarding the use of human embryos. The potential of iPS cells is enormous and the iPS stratagem has been exponentially growing, and numerous iPS lines have been generated. Additionally, several iPS cell derived somatic cell types are being evaluated for regenerative medicine and pharmaceutical application.
Many obstacles remain before their medical and pharmaceutical applications can be fully realized. The hurdle of teratoma formation leads the transplantation stratagem to choose terminally differentiated cells and/or committed progenitor cells over primary iPS cells. However, the differentiation of HCs walks through a complex spatial and temporal pathway, where multiple interplays of various factors at a highly organized sequence may be essential to a fully committed HC. This complexity discourages the guarantee of a pure population of HCs, because the orchestrated differentiation usually requires cross-talk of signals from the co-differentiating neighbors, similar to the patterning in vivo. Thus, isolation of the desired differentiation derivative is required for obtaining a pure cell population. A better understanding of the signaling pathways guiding the development and differentiation of iPS cells will enable us to design a step-wise procedure employing a “correct” mixture of regulatory factors to achieve high-yield differentiation. The advancement of iPS techniques and the ability of manipulating HC differentiation may eventually enable us to design a biological “implants.” Such implants may precisely reverse the degeneration remaining HCs or supporting cells in the deaf ear.
Other obstacles must be addressed before application in clinical therapy. Among these are; 1) identification of the aberrant reprogramming, which is a co-product during iPS cell preparation. Since, it may result in impaired ability to differentiate and may increase the risk of immature teratoma formation after directed differentiation. 2) The promised silencing of transgene integration into the iPS cell genome is another concern. Reactivation or leaky expression of such transgenes (e.g. c-Myc) could lead to tumor genesis, creating a greater risk of immature teratoma formation [45
2.3 Olfactory progenitors
The olfactory neuroepithelium in the mammalian nervous system is capable of generating new olfactory receptor neurons periodically throughout adult life, as well as the capacity to proliferate in response to acute injury. The unique proliferative feature results from the presence of multipotent stem cells in the olfactory neuroepithelium[46
]. An in vitro
differentiation of adult mouse olfactory precursor cells into HCs has been reported. These hair cell-like cells provide a potential autotransplantation therapy for hearing loss [47
]. Precursor cells from mouse olfactory neuroepithelium were sphere-forming, and showed proliferative capacity. After co-culturing with cochlear cells and/or cochlear supernatant, the olfactory progenitor cells expressed several HC markers, including myosin VIIa, calretinin, and espin. These results demonstrated for the first time that adult olfactory precursor cells could differentiate into hair cell-like cells. Thus, olfactory neuroepithelium offers an abundant and easily accessible source of adult stem cells, which could be a potential autotransplantation therapy for hearing loss. However, the functional potential of this source remains to be addressed.
2.4 Bone marrow derived cells
Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages, and have long been evaluated in clinical practice. An in vitro
overexpression of the prosensory transcription factor, Math1, will induce MSC into inner ear sensory cells which expressed myosin VIIa, espin, Brn3c, p27Kip, and jagged2. Some of the daughter cells demonstrated F-actin-positive protrusions, a structural analog of HC stereociliary bundles. Hair cell markers were also induced in culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event [48
]. An in vivo
evaluation to test its restorative concept in inner ear repair, demonstrated the homing capability of bone marrow-derived cell (BMDC) to the deafened cochlea. However, the spontaneous transdifferentiation to any cochlear cell type after acoustic trauma was not observed [49
]. These pilot studies suggest that pre-differentiation of bone marrow derived cells is required to achieve the possible hearing rehabilitation.
2.5 Stem Cells from Amniotic Fluid and Placenta
Since recent report has demonstrated the presence of stem cells with multiple potential in the amniotic fluid and placenta, they are now recognized as potential sources of stem cell therapy. Amniocentesis and chorionic villus sampling are widely accepted methods for prenatal diagnosis. Therefore, no ethical concerns will be aroused if embryonic and fetal stem cells would be taken from amniotic fluid and placenta before or at birth. Isolated human and rodent amniotic fluid-derived stem (AFS) cells express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders. Interestingly, AFS cells are broadly multipotent. They can differentiate into cell types representing each germ layer, including neuronal, osteogenic, and hepatic phenotypes [50
]. The demonstrated neuronal lineage indicates that AFS could be used for repopulation of inner ear sensory cells. Meanwhile, banking of these stem cells may provide a convenient source both for autologous therapy in later life and for matching of histocompatible donor cells with recipients.