Stem Cell Therapy

Background

One of the greatest challenges in the treatment of inner-ear disorders is to find a cure for the hearing loss that is caused by the loss of cochlear hair cells or spiral ganglion neurons. The recent discovery of stem cells in the adult inner ear that are capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens.

Cell-replacement therapy with stem cells has the potential to have a massive impact on human health during the coming decades. The first targets for therapeutic stem-cell applications are degenerative ailments, such as heart disease, diabetes, Parkinson’s disease and other neurodegenerative disorders. The initial results using stem-cell-based generation of replacement cells for these disorders indicate that stem cells can be developed into highly specialized cell types and that these new cells can function in animal models, even improving the underlying organ function. There are three principal sources for stem cells that have been used to (re-)generate organ-specific cell types: ES cells, stem cells that are isolated from the organ to be generated and stem cells from other organs. Consequently, the regeneration of lost hair cells can, theoretically, involve ES cells, inner-ear stem cells or stem cells from brain, skin or the hematopoietic system. [1]

Stem Cells for the Treatment of Degenerative Diseases

Hair cells from embryonic stem (ES) cells

ES cells are derived from the inner cell mass of the blastocyst. Because they are the precursors for all other embryonic cells, ES cells have the greatest capacity for differentiation into multiple cell types, which is termed pluripotency. ES cells also have the capacity for self renewal and can, therefore, be expanded to large numbers. The generation of specific cell types by directing ES-cell differentiation hypothetically offers an extensive resource for developing clinical applications to replace diseased or injured cells.  Recently, inner-ear progenitors have been generated from murine ES cells in vitro [2]. These progenitors express a set of marker genes that identify them as cells in the lineage of the hair cells, because these markers can only be found in this specific combination in the developing inner ear. After differentiation in vitro, a subpopulation of the ES-cell-derived progenitors exhibited a hair-cell phenotype, as revealed by the expression of characteristic markers such as the transcription factors Math1 (murine atonal homologue 1) and Brn3.1, which are important for the generation of, and for maintaining the maturation of, hair cells [3]. Expression of these transcriptional key regulators was accompanied by the upregulation of structural hair-cell proteins, such as the unconventional myosin VIIA, parvalbumin 3, and espin.

The implantation of genetically labeled ES-cell-derived inner-ear progenitors into the inner ear of chicken embryos and following their fate through early otic development showed that engrafted cells initiated the expression of hair-cell markers when situated in developing inner-ear sensory epithelia. Progenitor-derived cells that were found elsewhere in the inner ear did not express hair cell markers. Consequently, it has been hypothesized that grafted murine ES-cell-derived inner-ear progenitor cells can respond to local cues that control (hair) cell-type specification in the developing chicken inner ear [3]. Although the developing avian inner-ear sensory epithelia are different from injured or diseased mammalian organ of Corti or vestibular hair-cell-bearing epithelia, these results are the first successful approach using ES cells to generate hair cells in vivo.

Hair cells from adult stem cells

Stem cells have been isolated and propagated from many adult organs, including the brain, bone marrow, muscle, heart, skin, eye and, recently, from the inner ear [4]. Neural stem cells, which have the ability to differentiate into many neuronal cell types, have been successfully grafted into the drug-injured mouse inner ear; the cells survived for several weeks and expressed markers of mature cell types, including glia, neurons and hair cells, albeit not in the cochlea [5]. Comparison of the in vitro potential of adult neural stem cells with stem cells from the inner ear of adult mice revealed two substantial differences in the potential of the cells to differentiate into hair-cell-marker-positive cells. First, the upregulation of hair-cell markers was readily observed in 10% of all cells that were differentiated from innerear- derived stem cells in vitro, whereas adult neural stem cells that were isolated from the forebrain rarely (,0.1%) gave rise to hair-cell-marker-positive cells in this assay. Second, inner-ear stem cells appeared to differentiate more completely into hair cells than the neural stem-cell derivatives. This became apparent by the formation of hair-bundle-like structures that were immunopositive for specific stereociliary markers. In vitro inner-ear stem-cell-derived cells, after transplantation into a developing chicken inner ear, upregulate hair-cellspecific markers in a similar manner to grafted ES-cellderivatives.

Adult inner-ear stem cells reside in the sensory epithelium of the utricle and are a plausible candidate for the progenitor cells that have been postulated as the source of hair-cell regeneration in the damaged utricular sensory epithelium. Inner-ear stem cells are pluripotent because they can develop into many other cell types outside of the inner ear that are derived from either ectodermal, endodermal or mesodermal germ layers. The defining stem-cell feature of inner-ear stem cells is their high proliferative capacity, which makes it possible to isolate these cells in the form of clonal floating colonies or spheres. Proliferation potential is crucial to developing treatment strategies for hearing loss, because propagation of these cells might become the foundation of a replacement strategy for human inner-ear cells.

Do hair cells that are generated from stem cells follow the native developmental program?

The variety of cellular interactions that have roles during the complex development and morphogenesis of the inner ear are gradually being unraveled [6], although the signaling events that lead to the specification of individual inner-ear cell types are still largely unknown. Fortunately, markers for specific subpopulations of cells within the developing otic vesicle and its vicinity have recently been identified. Such markers enable subpopulations of cells to be followed through development, and the labeling of specific cell populations provides further insight, such as rapidly proliferating cells or cells that are undergoing apoptotic cell death [7].

One of the earliest markers to appear during the development of the ear is the paired-box transcription factor Pax2, which is expressed in all otic placodal cells (Figure 1a and 1b). After invagination, Pax2-expressing cells are predominantly localized in the ventral part of the otic vesicle in mice and in medio-ventral regions of the chicken otic vesicle [8]. Pax2 is expressed in proliferating progenitor cells of the presumptive inner-ear sensory patches and is downregulated in early differentiating hair cells, but maintained, albeit decreasingly, in early supporting cells. Robust expression of Pax2 is detectable in populations of progenitor cells that are generated from inner-ear stem cells and in presumptive otic placode-like cell types that are obtained from ES cells by selection with a combination of epidermal growth factor and insulin-like growth factor 1 (Figure 1c).

The presumptive sensory patches of the inner ear express a defining combination of markers that include the signaling proteins BMP4 and BMP7, the Notch-ligand Jagged-1 and the cell-cycle modulator p27Kip1 (Figure 1d–f). Concurrently, early sensory patches lack the expression of markers for differentiated or differentiating cell types, such as the hair-cell markers Math1, Brn3.1, myosin VIIA and espin. This temporal expression pattern of markers is also reflected in stem-cell-derived inner-ear progenitor cells. Hair-cell differentiation from innerear-derived stem cells was apparent by the substantial upregulation of marker genes for hair cells and by the appearance of cytomorphological specializations, in particular, filamentous actin-rich protrusions that display strong immunoreactivity for the hair-bundle marker espin (Figure 1g–k). Cells that displayed hair-bundle-like structures emerged almost exclusively on top of or closely surrounded by large cells that expressed markers for inner-ear supporting cells [2]. In differentiating cell populations that are generated from ES-cell-derived progenitors, hair-cell-like cells are rarely found in association with large supporting-cell-like cells. The in vitro generation of morphologically mature hair cells might, therefore, require intimate contact with cells that substitute for inner-ear supporting cells, as is the case in vivo, suggesting that the generation of functionally mature hair cells in vitro requires the co-generation of accessory cell types.

Panel (a), (d), (g), and (i) in figure 1 shows the schematic illustration of inner-ear sensory cell development: (a) otic placode stage, (d) early sensory epithelium, (g) early differentiating hair and supporting cells and (i) maturing sensory epithelium. A selection of marker genes that are expressed in developing sensory epithelia is indicated. (b) Labeling of the chicken otic placode with antibody to Pax-2 (red). (c) Inner ear progenitors, selected from embryonic stem (ES) cells, express Pax-2 protein (red). (e) In situ hybridization with a probe to chicken serrate-1 reveals the expression of the avian jagged homologue in cochlear (arrow) and vestibular (arrowhead) sensory epithelia. (f) Reverse-transcriptase PCR analysis reveals the expression of early sensory epithelium markers in inner-ear progenitor cells (IEPG) selected from ES cells. (h) Nuclear expression of Math1 (green) and cytoplasmic immunoreactivity for the hair-cell marker myosin VIIA (red) in a differentiating hair-cell-like cell derived from ES cells. (j) Myosin VIIA (red) and Math1 (green) expression in hair cells of a late embryonic section of the murine organ of Corti. Cell nuclei are visualized in blue. (k) Hair-bundle-like protrusion, labeled with red espin immunofluorescence, of a hair cell-like cell expressing Math1 that was generated from adult inner-ear stem cells (green).

Figure 1 Hair cell development and hair cell generation from stem cells

Reference

[1] Huawei Li, C. Eduardo Corrales, Albert Edge and Stefan Heller, "stem cells as therapy for hearing loss", Trends  in Molecular Medicine Vol.10 No.7 July 2004

[2] Li,H.et al. "Generation of hair cells by stepwise differentiation of embryonic stem cells". Proc. Natl. Acad. Sci. U. S. A. 100, 13495–13500, 2003

[3] Xiang, M. et al. "Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development". Proc. Natl. Acad. Sci. U. S. A. 94, 9445–9450, 1997

[4] Li,H. et al. "Pluripotent stem cells from the adult mouse inner ear". Nat. Med. 9, 1293–1299, 2003

[5] Tateya, I. et al. "Fate of neural stem cells grafted into injured inner ears of mice". Neuroreport 14, 1677–1681,  2003

[6] Zine, A. "Molecular mechanisms that regulate auditory hair cell differentiation in the mammalian cochlea". Mol. Neurobiol. 27, 223–238, 2003

[7] Lang, H. et al. "Cell proliferation and cell death in the developing chick inner ear: spatial and temporal patterns". J. Comp. Neurol. 417,
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[8] Lawoko-Kerali, G. et al. "Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear". J. Comp. Neurol. 442, 378–391, 2002