Учебники / Hair Cell Regeneration, Repair, and Protection Salvi 2008

142 E.C. Oesterle and J.S. Stone

by a concomitant growth of each otolithic organ over much of the lives of these animals (Platt 1977; Corwin 1981, 1983; Popper and Hoxter 1984). In contrast, the area of the utricular epithelium of birds ceases to expand shortly after hatching (Jørgensen 1991; Goodyear et al. 1999), although new HCs continue to be produced at a slow rate throughout this tissue and in other vestibular epithelia postembryonically (Jørgensen and Mathiesen 1988; Roberson et al. 1992). Because HCs in avian vestibular organs die spontaneously at a steady rate (Jørgensen 1991; Kil et al. 1997), it is assumed that postembryonic HC production serves to replace dead HCs and to maintain a relatively constant HC number. In vestibular and lateral line sensory organs of mature nonmammalian vertebrates, rates of HC production are upregulated in response to experimentally induced HC loss (Weisleder and Rubel 1992; Baird et al. 1993; Kil et al. 1997; Williams and Holder 2000; Avallone et al. 2003; Harris et al. 2003).

In the auditory sensory epithelium (SE) of nonmammals, normal HC and SC production appears to be largely confined to embryogenesis (Katayama and Corwin 1989, 1993). In the auditory SE (basilar papilla) of mature birds, only negligible cell division is detectable, and no new HCs are added (Corwin and Cotanche 1988; Ryals and Rubel 1988; Oesterle and Rubel 1993). However, experimental induction of HC loss leads to a significant upregulation in cell division and to the production of a full set of replacement HCs (Cotanche 1987a; Cruz et al. 1987; Corwin and Cotanche 1988; Ryals and Rubel 1988), assuming epithelial trauma is not too severe (see Cotanche et al. 1995; Ding-Pfennigdorff et al. 1998).

1.2 Mammals

In contrast to nonmammalian species, in mammals HC production ceases during the late embryonic period (Ruben 1967). Cell division does not occur spontaneously in mature mammalian auditory or vestibular SE (Roberson and Rubel 1994; Kuntz and Oesterle 1998a). No new auditory (organ of Corti) HCs are produced, either under normal conditions or after experimentally induced HC lesions (Sobkowicz et al. 1992; Roberson and Rubel 1994; Chardin and Romand 1995). However, rare mitotic activity is triggered in vestibular SE (Warchol et al. 1993; Lambert 1994; Rubel et al. 1995; Li and Forge 1997; Kuntz and Oesterle 1998a). Recent investigations have begun to explore the potential for inducing mitotic HC replacement in mammalian species via the addition of mitogenic growth factors to cultured mammalian tissue (Lambert 1994; Yamashita and Oesterle 1995) and to intact animals (Kuntz and Oesterle 1998a; Kopke et al. 2001) and by disrupting normal gene function (Chen and Segil 1999; Löwenheim et al. 1999; Mantela et al. 2005; Sage et al. 2005). Investigations have also begun to explore HC production in mammals by a nonmitotic process, through either repair or direct conversion of another cell type (e.g., supporting cells) into HCs (Zheng et al. 2000; Kawamoto et al. 2003; Izumikawa et al. 2005).

At this point, the following critical questions related to mammalian HC regeneration must be explored further: (1) Are cells with the potential to form new

HCs, either mitotically or nonmitotically, retained in the mature mammalian SE? (2) If so, how are these cells prevented from dividing after HC damage?

(3) Can such cells be triggered to generate new HCs using a noninvasive, harmless method? In this chapter, mechanisms used by nonmammalian species to regenerate HCs after birth are discussed, with a focus on factors regulating SE proliferation and differentiation. Then, the mammalian response to HC trauma is discussed, and currently promising approaches toward triggering replacement of mammalian HCs are considered.

2. Hair Cell Regeneration in Nonmammalian Species

Because most of the research on regeneration of auditory HCs in nonmammalian species has been conducted in birds, most of the discussion in this section focuses on studies in birds. Research on other animals is discussed as appropriate.

2.1 Cellular Proliferation

As discussed in the preceding text, mitotic activity in the SE gives rise to new HCs in many mature nonmammalian animals. Several studies have addressed the identity of progenitor cells in this regenerative process and have examined the timing, magnitude, and cellular/molecular events associated with progenitor cell division to gain insight into potential regulatory mechanisms used by species capable of mitotic HC regeneration. These studies are the focus of the next section.

2.1.1 Progenitor Cells: Identity, Location, and Behavior

All HC epithelia have two primary cell types: sensory HCs and nonsensory supporting cells (SCs) (Fig. 5.1). Supporting cells are interposed between HCs. While the cell bodies of HCs are confined to the lumenal surface of the SE, the somata of all SCs appear to extend from the lumen to the basal lamina. The nucleus of most SCs is located in the basal (adlumenal) half of the epithelium. For many years, SCs were suspected to serve as progenitors to new HCs during postembryonic HC production in mature nonmammalian vertebrates. The earliest evidence to support this hypothesis emerged from studies that demonstrated new SCs are also formed by mitosis, under normal and damaged conditions (Corwin 1981; Cotanche 1987a; Cruz et al. 1987; Corwin and Cotanche 1988; Ryals and Rubel 1988; Girod et al. 1989; Presson and Popper 1990). However, it was not until a decade ago that SCs were “caught in the act” of dividing, and their progeny were shown to go on to form new HCs and SCs. These observations were first made directly, after laser ablation of HCs in the lateral line end organs in live salamanders (Balak et al. 1990; Jones and Corwin 1993). Microscopic timelapse imaging allowed investigators to determine that SC nuclei migrate from the basal compartment of the epithelium to the lumenal surface, where they undergo

mitotic division. Analysis of SE from birds and fish also showed that SCs are the first cells to undergo DNA synthesis after HC damage, with some of the resulting progeny differentiating into HCs (Presson and Popper 1990; Raphael 1992; Hashino and Salvi 1993; Stone and Cotanche 1994; Tsue et al. 1994a; Warchol and Corwin 1996; Fig. 5.2A). Epithelial cells lining the SE called hyaline cells

Figure 5.2. Temporal progression of cellular events associated with hair cell regeneration in the chicken auditory epithelium (basilar papilla). This schematic depicts the temporal progression of cellular events involved in the two known modes of nonmammalian HC regeneration in the chicken basilar papilla: mitotic regeneration (A) and direct transdifferentiation (B). Mature and regenerating hair cells (HCs) are depicted with darker cytoplasm than the supporting cells (SCs). In both diagrams, events progress temporally from left to right, with the normal epithelium shown on the left, the damaged and regenerating epithelium in the middle, and the fully repaired epithelium on the right. Arrows point to damaged HCs in the process of being extruded into the lumenal fluid. Asterisks indicate regenerated HCs with elongated morphology. For mitotic regeneration (A), the stages of the cell cycle are depicted as G1 (gap1), S (DNA synthesis), G2 (gap2), and M (mitosis). BL, Basal lamina. (Reprinted from Bermingham-McDonogh O, Rubel EW. Hair cell regeneration: winging our way towards a sound future. Curr Opin Neurobiol 13:119–126, Copyright 2003, with permission from Elsevier.)

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are induced by severe noise trauma to undergo cell division and to migrate into the damaged SE. However, unlike SCs, hyaline cells do not appear to be capable of giving rise to new HCs (Cotanche et al. 1995).

Over the nearly two decades after the identification of SCs as progenitor cells, substantial work was done to characterize progenitor cell behavior after HC damage, primarily in avian species (reviewed in Stone and Rubel 2000b). Nonetheless, the following questions remain unanswered: (1) Are distinct subsets of SCs specialized to serve as HC progenitors? (2) Do all dividing cells give rise to HCs, or are there other cell lineages present in inner ear SE? (3) How are HC progenitors renewed during regeneration; is there a tissue stem cell found in inner ear SE?

Adult tissue stem cells are self-renewing progenitor cells that can generate one or more specialized cell types. They are found in many adult tissues, including epithelial tissues (e.g., olfactory epithelium, epidermis, crypts of the intestinal mucosa), muscle, the hematotopoetic system, and brain (Weissman et al. 2001; Wagers and Weissman 2004). Adult stem cell fate is controlled by intrinsic factors and extrinsic factors, the latter of which are provided by the cell’s specialized microenvironment (the stem cell niche; for review see Watt and Hogan 2000).

In other sensory systems capable of regeneration, nonsensory cells are highly divergent, and distinct cell lineages appear to be present (e.g., retina: Fischer and Reh 2000, 2001; olfactory epithelium: reviewed in Schwob 2002). These lineages include stem cells, intermediate progenitors, and terminally mitotic cells. Stem cells are founder progenitor cells; they give rise to a range of cell types within a tissue, and they renew themselves when they divide, thereby ensuring the capability of future tissue repair. Intermediate progenitor cells are generated by stem cell divisions, but unlike stem cells, they have a limited potential. In a sensory tissue, intermediate progenitors typically give rise to terminally mitotic cells, such as sensory receptors. An excellent example of a neuroepithelial cell lineage is the mammalian olfactory epithelium. Olfactory receptor neurons are regenerated in mature mammals in response to several types of trauma (reviewed in Murray and Calof 1999; Schwob 2002). Careful analysis has demonstrated that, in addition to olfactory neurons, at least five types of nonsensory cells exist in the olfactory epithelium, each with distinct morphological and molecular properties. Sustentacular cells, microvillar cells, and Bowman’s gland/duct cells all span the width of the olfactory epithelium. In contrast, the cell bodies of horizontal basal cells and globose basal cells reside near the basal lamina. When olfactory neurons are selectively killed, only globose basal cells show a large increase in mitotic activity (Schwartz Levey et al. 1991). Transplantation of globose basal cells into an olfactory epithelium where more than one cell type has been injured shows that these cells can give rise to a large range of cell types, including sustentacular cells, globose basal cells, and neurons (Goldstein et al. 1998). This finding demonstrates that some globose basal cells have properties of multipotent stem cells. Analyses of cell fate during regeneration show that several cell lineages exist in the olfactory

epithelium, and some are more restricted than others. For example, progenitors that give rise to Bowman’s gland/duct cells do not appear to generate neurons (Huard et al. 1998). In the neuronal lineage, globose basal cells with stem cell properties are few in number (approximately 1/3600; Mumm et al. 1996) and appear to divide at a slow rate. In the process, they renew themselves and form intermediate progenitors (Calof and Chikaraishi 1989), which are more predominant and divide at a faster rate. The progeny of intermediate progenitors eventually undergo terminal differentiation, as neurons or nonsensory cells.

Knowledge of subsets of nonsensory cells in the olfactory epithelium has enabled relatively rapid progress in identifying signals that regulate the behavior of distinct cell types (reviewed in Beites et al. 2005). In contrast, little is known about SC specialization or cell lineages in mature inner ear epithelia. The presence of a self-renewing progenitor (stem) cell among the SC population in the avian basilar papilla is supported by several studies (reviewed in Parker and Cotanche 2004). The basilar papilla of the Belgian Waterslager canary undergoes spontaneous HC death after birth and appears to continually regenerate new HCs and SCs (Gleich et al. 1994, 1997). In starlings and quail, auditory HCs are regenerated after two separate HC injuries, suggesting progenitor cells are renewed following the first insult (Niemiec et al. 1994; Adler and Saunders 1995; Marean et al. 1995). In the damaged chicken basilar papilla, between 1% and 4% of SCs divide twice during the course of recovery from drug-induced HC loss (Stone et al. 1999), suggesting a capacity for self-renewal in at least some SCs. Some progenitor recycling also appears to occur in the chick basilar papilla after noise damage (Stone and Cotanche 1994). Further, some mitotic events in the damaged basilar papilla are asymmetric in nature, producing a HC and a SC (Stone and Rubel 2000a). Asymmetric division leading to the replenishment of a cell with proliferative capacity is one hallmark of stem cells.

Data from nonmammalian vestibular epithelia support the presence of specialized stem cells in that tissue as well. For example, while growth of the chicken utricle slows considerably over time after hatching (Jørgensen 1991; Goodyear et al. 1999), progenitor cell division proceeds at a substantial rate into adulthood (Roberson et al. 1992; Kil et al. 1997). The presence of a stem cell is assumed, as the progenitor population must be replenished in order to maintain life-long mitotic activity. Interestingly, though, it has been difficult to document progenitor cell recycling in the undamaged chicken utricle, perhaps due to a slow cycling rate (Stone et al. 1999; Wilkins et al. 1999). In contrast, in the undamaged fish saccule, as many as 70% of the progenitor cells reenter the cell cycle within days of completing it (Wilkins et al. 1999). Removal of rapidly dividing cells has been achieved by treatment of organs in vitro with cytosine arabinoside (Ara-C), an antineoplastic drug that causes death in cells entering S phase in many tissues, including the brain (Jung et al. 2004). Ara-C treatment in the fish saccule causes the loss of dividing cells and the recruitment of another group of SCs into the cell cycle (Presson et al. 1995). Based on studies of cell lineages in other systems, this finding implies the presence of a rapidly dividing

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intermediate progenitor (susceptible to Ara-C) in the fish saccule along with a slowly dividing or quiescent stem cell (resistant to Ara-C; reviewed in Hall and Watt 1989).

While several features distinguish HCs from SCs in the nonmammalian SE (e.g., Tanaka and Smith 1978; Ginzberg and Gilula 1979), very few morphological, molecular, or behavioral features clearly delineate subsets of SCs. This is in contrast to the mammalian organ of Corti, which contains several subtypes of SCs that are structurally and chemically distinct. In the late embryonic chicken basilar papilla, two different SC morphologies have been documented: cells with thick apical and basal processes and a centrally located nucleus, and cells with thin, delicate processes and an adlumenally located nucleus (Fekete et al. 1998). It is not known if these subtypes are functionally distinct or if they are retained in mature chickens. Electron microscopic studies of SCs in fish and chickens have revealed no marked systematic differences among SCs at the ultrastructural level (Presson et al. 1996; L.G. Duckert, unpublished observations). However, small morphological and ultrastructural differences have been reported for the several rows of SCs that line the abneural (inferior) edge of the papilla in comparison to SCs that are situated between HCs (Oesterle et al. 1992). Furthermore, nuclei of S-phase SCs in the fish vestibular epithelium display a different shape and position than quiescent SCs (Presson et al. 1996). While some molecular markers of SCs have been characterized (e.g., cytokeratins: Stone et al. 1996; receptorlike protein tyrosine phosphatase [RPTP]: Kruger et al. 1999; tectorins: Goodyear and Richardson 2002), only a handful of markers show systematic variability across the quiescent SC population. For example, mRNA for fibroblast growth factor receptor-3 (Bermingham-McDonogh et al. 2001) is present in high levels in most SCs, but it is absent from those located along the neural (superior) edge of the basilar papilla. In addition, proliferating cell nuclear antigen (PCNA) is immunologically detectable in 3% of SC nuclei, although no overt pattern of spatial distribution is evident (Bhave et al. 1995). Further, expression of the homeobox transcription factor, cProx1, reveals molecular diversity among quiescent SCs and among dividing progenitor cells in the basilar papilla (Stone et al. 2004). The relevance of this variation in expression of these different genes among SCs has not been determined. However, all three genes have been implicated in cell cycle regulation in other tissues/species (Wigle et al. 1999; Li and Vaessin 2000; Dyer et al. 2003; Hong and Chakravarti 2003; InglisBroadgate et al. 2005). Assuming these proteins play similar roles in the chicken basilar papilla, these findings support the existence of progenitor populations with different potentials for cell division. Alternatively, these differences may reflect stochastic variability in gene expression across the SC population.

For the remainder of this chapter, HC progenitors are presumed to reside among the SC population in nonmammals, so the terms SC and progenitor cell are used interchangeably, with some distinctions made in context. As discussed in Section 3.1.1, several lines of evidence suggest that tissue stem cells are intrinsic to mammalian vestibular epithelia, albeit in limited numbers and in a quiescent or restricted state. Further characterization of nonmammalian stem/progenitor

cells would provide information for comparative studies across nonmammals and mammals that may shed light on why mitotic activity and HC regeneration are so highly attenuated in mammals.

2.1.2 Proliferation Signals

In nonmammals, mitotic activity in progenitor cells is robust in response to damage. For example, in the chicken basilar papilla, hundreds of SCs incorporate the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) after a single bolus of gentamicin is delivered intraperitoneally; over the course of recovery, thousands of SCs appear to divide (Stone et al. 1999). The section that follows discusses studies that have laid the groundwork for our current understanding of the cellular processes leading to the entry of HC progenitors into the cell cycle and of the molecules associated with these processes.

2.1.2.1 Mitotic Activity Is Dependent on Location, Timing, and Magnitude of Hair Cell Death

Mitotic activity among HC progenitors is spatially, temporally, and quantitatively correlated with HC death. This is best illustrated by experimental studies in which location, timing, and amount of HC death are known entities, such as after noise-, ototoxic drug-, or laser-induced traumas to the auditory epithelium (discussed in Forge and Van De Water, Chapter 6). Exposure to pure-tone or narrow-band noise stimuli leads to HC lesions in the tonotopically corresponding region of the auditory epithelium. The proportion of HCs (and SCs in extreme cases) that are killed within this region depends on the intensity and duration of acoustic overstimulation. Administration of ototoxic drugs, such as aminoglycoside antibiotics (e.g., gentamicin, neomycin), causes substantial HC death to the proximal, high-frequency portion of the auditory epithelium (reviewed in Cotanche 1999; Forge and Schacht 2000). Prolonged drug exposure, or increased drug dosages, leads to expansion of the damaged region toward the distal, lowfrequency end of the organ. Single-dose regimens lead to smaller lesions that are generated in a shorter amount of time, and these have proven effective for studying the timing of cellular events that are presumably interdependent, such as HC death and cell division, discussed later (Bhave et al. 1995; Janas et al. 1995; Roberson et al. 1996). Combinations of noise exposure and drug treatment have profound effects on HCs, causing damage and/or death across nearly the entire auditory organ (e.g., Bone and Ryan 1978; Woolley and Rubel 1999). Another method of killing HCs, laser ablation, is directed at single HCs or groups of HCs in vivo (Jones and Corwin 1993) or in vitro (Warchol and Corwin 1996).

After each method of HC injury, SC proliferation is limited to regions of severe HC trauma and/or HC death (Corwin and Cotanche 1988; Ryals and Rubel 1988; Hashino and Salvi 1993, Hashino et al. 1995; Stone and Cotanche 1994; Warchol and Corwin 1996; Fig. 5.2A). Interestingly, two studies in the chicken basilar papilla show that progenitors residing outside the area of overt HC damage show a limited change in cell cycle status, even though HCs surrounding them

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do not display clear signs of damage (Bhave et al. 1995; Sliwinska-Kowalska et al. 2000). As discussed in the preceding text, cell division is very rare in the undamaged basilar papilla. This feature is reflected by the fact, that in the undamaged papilla, most SCs (62%) express an antigen characteristic of growth arrest (the G0 marker, statin), whereas 0–3% of SCs express proliferating cell marker (PCNA), an antigen characteristic of G1 and later stages of the cell cycle (Fig. 5.3). Short treatments with the ototoxin gentamicin led to complete HC loss in the proximal (high-frequency) end of the basilar papilla. In response to gentamicin treatment, statin and PCNA expression are significantly altered, with statin expression decreasing and PCNA expression increasing. Remarkably, these changes occur in SC nuclei throughout the basilar papilla, including the distal undamaged area, demonstrating widespread SC transit from growth arrest (G0) to the gap 1 phase of the cell cycle (G1). A similar response was seen in the basilar papilla after exposure to wide-band noise; PCNA immunoreactivity increased in both damaged and undamaged regions (Sliwinska-Kowalska et al. 2000). Importantly, though, progression of SCs to DNA synthesis (S phase) and later stages of the cell cycle only occurred in areas of complete HC loss. These studies demonstrate that, like many other tissues including the central nervous system (Galderisi et al. 2003), cell cycle progression in avian SCs depends on multiple highly controlled signals. They suggest that, after HC damage, signals that trigger SC transit from G0 to G1 are altered throughout the tissue, while signals directing SCs to enter S phase are limited to areas of marked HC damage. Future studies should strive to identify molecules that act regionally to promote each step in cell cycle progression.

The time course of cell division after experimental HC injury has been examined most extensively in chickens (Raphael 1992; Hashino and Salvi 1993; Stone and Cotanche 1994; Bhave et al. 1995; Hashino et al. 1995; Warchol and Corwin 1996; Stone et al. 1999; Roberson and Cotanche 2000). In the basilar papilla, entry of progenitor cells into S phase is first seen between18 and 24 hours after the onset of acoustic overstimulation, drug treatment, or laser ablation. The reason for this delay is not known, but it is undoubtedly influenced by the timing of the signaling cascades directing progenitor cells to transit from growth arrest, their normal state (Oesterle and Rubel 1993), to S phase (discussed in more detail in Section 2.1.2.3). The trigger for this cascade appears to be the onset of HC damage because the latency period for SCs to reach S phase is similar in all damaging paradigms, regardless of the nature and duration of the damaging treatment (e.g., see Stone and Cotanche 1994; Warchol and Corwin 1996; Bhave et al. 1998). Perhaps the most dramatic demonstration of the dependence of progenitor cell cycle entry on the onset of HC damage is provided in a study by Hashino et al. (1995). Chronic treatment of chickens with the aminoglycoside kanamycin creates a HC lesion in the basilar papilla that spreads from proximal to distal over time (Hashino et al. 1991). Labeling of dividing cells with BrdU for short intervals at different periods relative to the start of drug exposure demonstrates that SCs in the proximal region, which are damaged initially, are the first to divide, and SC entry into the cell cycle progresses up the length of

Figure 5.3. Cell cycle progression in the chicken basilar papilla after gentamicin treatment. This figure depicts changes that occur with respect to cell cycle status among SCs in different regions of the damaged basilar papilla after gentamicin treatment. Data were derived from Bhave et al. (1995). Cell cycle status was assessed by immunolabeling for different cell cycle-related markers: statin, PCNA, and bromodeoxyuridine (BrdU). (A) Stage of the cell cycle where labeling for each marker is elevated. Stages of the cell cycle are depicted as G1 (gap1), S (DNA synthesis), G2 (gap2), and M (mitosis); G0 is growth arrest. Statin is elevated in cells in G0, but not in cycling cells. PCNA is elevated in cells in G1, S, and M phase, but not in cells in G0. BrdU labels cells in S phase and onward (G2, M, post-M). (B) Immunolabeling for hair cells and BrdU in the basal half of the epithelium of normal and damaged (3 days post-gentamicin) birds. In all panels, the basal tip of the epithelium is to the left, and the middle portion of the epithelium is to the right. The lines in the right-hand panels delineate the border of the HC lesion in the damaged basilar papilla. Note that no BrdU incorporation is seen in the normal epithelium, and that after damage, BrdU incorporation is confined to the region of complete hair cell loss. (C) Spatial patterns of hair cell loss and immunolabeling for BrdU, statin, and PCNA are shown for the epithelium at 3–5 days post-gentamicin. Each image is a full basilar papilla, with the proximal tip to the left and the distal tip to the right. Relative levels of hair cell loss and immunolabeling are indicated by the intensity of the gray label. Note the spatial correlation between hair cell loss and BrdU incorporation, but the lack of correlation between hair cell loss and statin/PCNA labeling.