Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

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14.1 Introduction

The retina is a unique sensory organ with a network of specialized neurons responsible for light perception and construction of visual images in the brain. Widespread damage of retinal neurons leads to irreversible blindness, for which new advances in stem cell research provide a hope for restoration of visual function in patients affected by retinal degenerative conditions.

Major retinal degenerative diseases that may potentially benefit from stem cell therapies include: age-related macular degeneration (AMD), which affects between 20 and 25 million people worldwide (Chopdar et al., 2003); proliferative diabetic retinopathy (PDR), which affects more than 35% of individuals after 20 years of diabetes (Kohner et al., 1998); other common retinal disorders such as end-stage glaucoma, retinitis pigmentosa (RP), proliferative vitreoretinopathy and inherited retinal diseases, which affect a large number of individuals during their productive life (Rosenberg and Sperazza, 2008).

Recent studies in the adult human eye have uncovered various sources of neural retinal progenitors, which under appropriate regenerative conditions

may potentially be used to promote retinal regeneration. Although the retina harbours these cells in adulthood, they appear to remain quiescent and there is no evidence that they re-enter the cell cycle or undergo neural differentiation following retinal injury. This brings us to speculate that the developed retina may provide an inhibitory environment for these cells to proliferate or differentiate in situ. However, unravelling the mechanisms that promote their quiescence in adulthood may help to identify factors to promote the growth and differentiation of these cells without the need for transplantation. Alternatively, retinal stem cells may potentially be expanded in vitro for autologous or syngeneic grafting, and this approach may require demanding and specialized resources. Understanding developmental pathways, cell requirements for in vivo expansion and survival, and the microenvironment in which progenitor cells are to be transplanted are essential for the development of successful cell-based therapies. To date, several methods have been used experimentally to deliver stem cells with limited success, but research in the field is rapidly expanding. Cell transplantation into the retina may require structural support, and new advances in the biomaterials field may lead to the development of appropriate scaffolds for cell delivery, which may promote the survival of transplanted cells and therefore facilitate the establishment of such treatments.

14.2Retinogenesis and stem cells in the adult human eye

The eye develops from three different types of embryonic tissue: the cornea and sclera develop from the mesoderm, the lens develops from the surface ectoderm and the retina and retinal pigmented epithelium (RPE) from the neural ectoderm (Graw, 1996). Although most of the studies in retinal neurogenesis have been performed in small species such as fish, amphibians, avians and rodents, there is a general consensus that similar developmental pathways occur in the human eye. Neural cell differentiation in the embryonic retina first occurs in the central optic cup, near the optic nerve head (Prada et al., 1991). This is followed by the migration of differentiating cells along the proximo-distal axis, i.e., in the direction of the central retina to the iris (GalliResta et al., 1997; Reese et al., 1995). During late retinogenesis, an increase in the proportion of postmitotic cells is observed and new cells are generated in a proliferative zone, the neuroblast layer, from where differentiating neurons migrate into laminating cell layers. Retinal ganglion cells are the first differentiated retinal neurons that emerge, followed in overlapping phases by horizontal cells, cone photoreceptors, amacrine cells, rode photoreceptors, bipolar cells and finally Müller glia (Cepko et al., 1996; Young, 1985).

Stem cells located at the margin of the neural retina immediately adjacent to the ciliary epithelium were first identified in fish and amphibians. This

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region, known as the ciliary marginal zone (CMZ), is known to harbour retinal progenitors responsible for the regeneration of neural retina in these species throughout life (Hollyfield, 1968; Raymond and Hitchcock, 1997). The presence of a region similar to the fish CMZ has also been shown in avians and small mammals during early postnatal life (Fischer and Reh, 2001; Ooto et al., 2004), and a similar anatomical region has now been described in the human eye (Bhatia et al., 2009). Another population of neural retinal progenitor cells has also been identified within the ciliary epithelium of the postnatal mammalian eye (Gu et al., 2007; Tropepe et al., 2000). However, much confusion has occurred with the understanding of the origin of these cells as a result of various reports giving different descriptions of the anatomical region from which ciliary epithelial cells with progenitor characteristics have been isolated from the mammalian eye. While reporting the presence of neural progenitors in the adult mammalian eye, various groups have failed to identify the anatomical provenance of these cells accurately. The first report that identified these cells in the mouse eye described them as ‘pigmented ciliary margin cells’ (Tropepe et al., 2000), while in the human eye they were reported to ‘derive from the pars plicata and pars plana of the ciliary margin’ (Coles et al., 2004). A more recent report described a ‘comparison of retinal progenitor cells isolated from the pars plana with a population of progenitors isolated from the ciliary body’ (MacNeil et al., 2007). It is therefore important to clarify that both the pars plana and the pars plicata are adjacent areas of the ciliary body (Bron et al., 1997), that the ciliary body is formed by two epithelial cell layers – one pigmented and the other non-pigmented (Bron et al., 1997) – and that the term ‘ciliary margin’ has traditionally been given to the marginal region of the neural retina, which is adjacent, yet different, to the ciliary body and which has been shown to harbour populations of neural progenitors in some species (Perron and Harris, 2000) (Fig. 14.1). Clarifying the fact that the ciliary body is not part of the neural retina may help us to understand the different nature of the various progenitor populations so far identified in the human eye. Interestingly, at the time of publication of this book, it has become clear that not only the anatomical origin of these so-called ‘ciliary margin’ cells has been incorrectly cited by many authors, but also the ‘stem cell nature’ of these cells in the adult eye has become incorrectly a dogma. Extensive evidence has now been presented that ciliary epithelial cells are not retinal stem cells (Cicero et al., 2009) but only ciliary epithelium that express pan-neuronal markers and does not form bona fide retinal neurons or glia in vivo or in vitro (Cicero et al., 2009). In this context, it might be important to clarify whether these properties of the ciliary epithelium vary within species and during different developmental stages.

During development, Müller glia and retinal neurons share a common progenitor that is multipotent at all stages of retinal histogenesis (Raymond

RPE

Pigmented epithellum

Non-pigmented epithellum

Ciliary

Laminated margin Pars plana Pars plicata retina

14.1 Diagram showing the anatomical localization of the ciliary body and the neural retina in the mammalian eye. The non-laminated ciliary marginal zone is present in fish and amphibians throughout life (Raymond and Hitchcock, 1997) and has also been observed in early postnatal life of avians and mammals (Fischer and Reh, 2001; Ooto et al., 2004).

and Hitchcock, 1997). This evidence derives from examination of the progeny of a single mouse retinal progenitor cell transfected with a retrovirus, which generated clones containing up to three types of neurons, while others contained a combination of neurons and Müller glia, Müller glia alone or a single type of neuron (Raymond and Hitchcock, 1997). Müller glia have been identified as a source of progenitor cells and retinal regeneration in the postnatal chick (Fischer and Reh, 2001). They have also been shown to proliferate and to produce some neuronal cell types (bipolar and rod photoreceptors) after neurotoxic injury to the adult rat retina (Ooto et al., 2004). Subsequent studies have unequivocally demonstrated that Müller glia exhibit neurogenic characteristics in the adult zebra fish (Raymond et al., 2006), and that these cells form the retinal stem cell niche, which is able to generate neurons after retinal injury in this species (Raymond et al., 2006). More recent investigations have shown that a population of Müller glial cells with stem cell characteristics is also found in the adult human retina (Bhatia et al., 2009; Lawrence et al., 2007) (Fig. 14.2), suggesting that humans may have some potential for neural retinal regeneration.

Retinal progenitor/stem cells can be isolated and induced to proliferate in vitro from the foetal and adult neural retina of the human eye using enzymatic dissociation methods (Kelley et al., 1995; Klassen et al., 2004; Lawrence et al., 2007). To promote their proliferation, retinal stem cells can be cultured in the presence of growth and differentiation factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF) (Kelley

Nestin

14.2 A population of Müller glia from the adult human retina exhibit neural stem cell characteristics. (a) A small proportion of Müller glia cells that expand across the width of the retina express markers of neural progenitors such as nestin (fluorescent label shown in white). (b), These cells can be isolated from the human neural retina and become spontaneously immortalized. In culture they show long processes that resemble Müller glia in situ (black arrow). (c), Müller stem cells cultured at low density in the presence of fibroblast growth factor 2 (FGF2) form neurospheres and express cyclin D, a marker of proliferating cells. (d) and (e) Neurospheres stain for nestin and cells contained within them express markers of differentiated retinal neurons such as rhodopsin (a photoreceptor marker, thin arrow) and Brn3 (a ganglion cell marker, open arrow showing nuclear staining). scale bars, 50 μm.

promotes multipotency of retinal progenitor cells (Marquardt and Gruss, 2002); (b) sonic hedgehog protein (Shh), which promotes proliferation and differentiation of progenitor cells into ganglion cells (Moshiri and Reh, 2004);

(c) Chx10, one of the earliest markers of the developing retina, which is required for retinal cell proliferation and formation of bipolar cells (Chen and Cepko, 2000); (d) basic helix–loop–helix (bHLH) transcription factors, such as Math 5 and NeuroD, which drive progenitor cells towards the ganglion cell and amacrine lineages, respectively (Ahmad et al., 1995); and (e) Sox2, a transcription factor found in early neurogenesis, which is downregulated as cells differentiate and migrate to the different retinal cell layers (Taranova et al., 2006). Expression of Sox2 has also been found to be an important marker of Müller stem cells in the adult human retina (Bhatia et al., 2009; Lawrence et al., 2007). Retinal stem cells that proliferate under the influence of growth factors may also be induced to differentiate into cells expressing markers of retinal neurons by modifications in culture conditions, such as the presence of extracellular matrix proteins and the addition of differentiation factors such as FGF, retinoic acid, insulin and the thyroid hormone T3 (Kelley et al., 1995; Klassen et al., 2004; Lawrence et al., 2007).

14.3Regeneration of neural retina

The first studies investigating the possibility of regenerating retina involved the transplantation of whole eyes to genetically eyeless salamanders (Harris, 1982). Implantation of peripheral nerves into adult rat retina (So and Aguayo, 1985), and grafting of embryonic rat retina into damaged adult rat retina have also been performed (Turner et al., 1986), but without success. Several investigations using various models of retinal degeneration have been developed; with various sources of stem cells used for retinal transplantation. These have included brain-derived stem cells (Young et al., 2000), embryonic retinal progenitor cells, ciliary epithelium and stem cells from the postnatal eye (Chacko et al., 2003), human embryonic stem cells (Banin et al., 2006), umbilical cord tissue cells and mesenchymal stem cells (Lund et al., 2007), bone marrow stem cells (Otani et al., 2004) and Müller stem cells (Lawrence et al., 2007). However, despite intensive research in the field, there is no evidence for widespread stem cell integration into the retina, long-term graft survival or complete restoration of visual function.

Neural progenitors obtained from the hippocampus have been shown to survive for very short periods of time after transplantation into the retina, and have failed to express markers of terminally differentiated retinal neurons, such as rhodopsin (Young et al., 2000). Lack of retinal marker expression has also been seen when other brain-derived precursor cell lines have been used for grafting into degenerating retina (Warfvinge et al., 2001). In contrast, progenitor cells obtained from the foetal retina have shown, in addition to

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good survival, expression of photoreceptor-specific markers after retinal transplantation. However, they have not integrated as well as hippocampalderived progenitor cells (Chacko et al., 2000). Damaged, dystrophic or degenerating retinae have been shown to promote the migration and integration of grafted retinal progenitors (Chacko et al., 2003; Mellough et al., 2004). These observations suggest that factors that regulate neuronal differentiation and synaptic connectivity during development might be reactivated by retinal degenerative processes (Sheen and Macklis, 1995).

Restoration of retinal function by transplanted stem cells requires the functional restoration of neural synapses, and the potential success of such therapies would depend on the ability of grafted cells to undergo neural differentiation and restoration of synaptic pathways within the host retina. On this basis it is more likely that retinal-specific stem cells from the adult eye that have undergone the developmental stages to become retinal neurons, may constitute more suitable candidates for cell-based therapies to restore retinal function than stem cells derived from embryos or other adult tissues.

At present, it is not known which conditions are needed to achieve optimal integration and long-term survival of transplanted stem cells. Whether better integration, neural differentiation and long-term graft survival may be obtained by transplantation of cell suspensions or cells supported by biomaterial scaffolds, or whether previously differentiated stem cells in vitro may functionally integrate better than non-differentiated cells, is not known. Experimental studies in a mouse model of retinal degeneration have suggested that efficient integration and differentiation of retinal progenitors into functional photoreceptor cells may be observed when specific neural precursors are transplanted (MacLaren et al., 2006). On this basis, in vitro differentiation of retinal stem cells may be necessary before transplantation and this may require intensive and careful laboratory procedures to protect their biological integrity and to avoid carcinogenesis. In addition to the type of stem cells potentially considered for cell-based therapies, the environment in which the cells are to be transplanted may need to be well thought-out, as abnormal deposition of extracellular matrix and the presence of pro-inflammatory cells that characterize retinal gliosis, may prevent the migration, differentiation and long-term survival of the grafts. Moreover, growth factors known to induce differentiation of neural stem cells during in vivo development could potentially be used as adjuvant therapies to induce neural differentiation and proliferation of adult retinal stem cells in situ. Following this line of research, recent evidence has shown that Shh, a protein that plays an important role in regulating neurogenesis during retinal development, and wingless-type protein-a (Wnt3a), a transcription factor important for neurogenesis, are able to induce in situ proliferation of Müller stem cells and generation of photoreceptors derived from these cells following neurotoxic damage or degeneration of mammalian retina (Osakada et al., 2007; Wan et al., 2007).