In vitro characterization of adult human retinal stem cells

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In vitro characterization of adult human retinal stem cells

Rebecca C. Frøen

Center for Eye Research / Department of Ophthalmology Oslo University Hospital and University of Oslo

PhD thesis 2017

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© Rebecca C. Frøen, 2018

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-241-8

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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1. Acknowledgements

This study was performed at Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo.

I wish to thank everyone who has contributed to this thesis. In particular I would like to thank my main supervisor, Morten C. Moe, for introducing me to the field, providing guidance and constructive criticism, and for encouraging me through all the ups and downs of working on this project. A special thanks to my co-supervisor Agate Noer without whom this thesis would not have been completed. I would also like to thank my two other co-supervisors Bjørn Nicolaissen and Iver A. Langmoen for sharing their insights on difficult topics. I am very grateful to Kristiane Haug Berg, for introducing me to all the practicalities of research and taking the time to teach me how everything in a lab works. Further I would like to thank the other members of our research group and all the other people who have contributed to this work. In particular I wish to mention Erik O. Johnsen, Aboulghassem Shahdadfar and Goran Petrovski. Finally, I would like to thank the members of the research group at Vilhelm Magnus Centre for Neurosurgical Research for fruitful collaboration and sharing of ideas.

Gratitude goes to my friends and family for providing me with their continued support and encouragement.

The project was financed by the Faculty of Medicine University of Oslo, Oslo University Hospital, the Blindemissionen IL and the Norwegian Association of the Blind and Partially Sighted.

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2. List of papers

Paper I

Moe M.C., Kolberg R.S., Sandberg C., Vik-Mo E., Olstorn H., Langmoen I.A., Nicolaissen B. A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain. Experimental Eye Research, 2009, Jan;88(1):30-8.

Paper II

Froen, R.C., Johnsen, E.O., Petrovski, G., Berenyi, E., Facsko, A., Berta, A., Nicolaissen, B., and Moe, M.C. Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells. Acta Ophthalmologica, 2011, Dec;89(8),e635-44.

Paper III

Johnsen, E.O., Froen, R.C., Albert, R., Omdal, B.K., Sarang, Z., Berta, A., Nicolaissen, B., Petrovski, G., and Moe, M.C. Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy. Experimental Eye Research, 2012, May;98:28-36.

Paper IV

Johnsen, E.O*., Froen, R.C*., Olstad, O.K., Nicolaissen, B., Petrovski, G., Moe, M.C., Noer, A. Proliferative cells isolated from the human peripheral retina only transiently upregulate key retinal markers upon induced differentiation. * Co-first authors. Current Eye Research, 2017, in press.

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3. Abbreviations

AMD age-related macular degeneration BMP bone morphogenetic protein

CB ciliary body

CE ciliary epithelium

Chx10 cation/H+ exchanger 10

CMZ ciliary marginal zone

CNS central nervous system

Crx cone-rod homeobox

Ct threshold cycle

Dcx doublecortin

ECM extracellular matrix EGF epidermal growth factor

FC fold change

FGF fibroblast growth factor

GAPDH glyceraldehyde-3-phosphate dehydrogenase GFAP glial fibrillary acidic protein

IHC immunohistochemistry

IPA ingenuity pathway analysis

IPE iris pigmented epithelium

iPSC induced pluripotent stem cell Map2 microtubule-associated protein 2

MITF melanogenesis associated transcription factor mRNA messenger ribonucleic acid

Nanog Nanog homeobox

NR neural retina

NRL neural retina leucine zipper

NSC neural stem cell

OCT optical coherence tomography Oct3/4 POU class 5 homeobox 1

Olig2 oligodendrocyte transcription factor 2

Pax6 paired box 6

PCR polymerase chain reaction

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8 PEDF pigment epithelium-derived factor

PR peripheral retina

PVR proliferative vitreoretinopathy

qRT-PCR quantitative reverse transcription polymerase chain reaction

RD retinal detachment

RHO rhodopsin

RMA robust microarray analysis

RPE65 retinoid isomerohydrolase/retinal pigment epithelium 65 rRNA ribosomal ribonucleic acid

RNA ribonucleic acid

RPC retinal progenitor cell

RPE retinal pigmented epithelium SEM scanning electron microscopy

siRNA short/small interfering ribonucleic acid

Sox2 SRY-box 2

SVZ subventricular zone

TEM transmission electron microscopy

TF transcription factor

TGFβ transforming growth factor β Vsx2 visual system homeobox 2

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4. Introduction

4.1 The eye

The eye is a complexly structured optical and sensory organ which facilitates vision. For an image to be captured by the brain, light must first travel through and be properly refractioned by the optical media of the eye – the tear film, cornea, lens and vitreous. In the retina, light is translated into electrical impulses that are processed and transmitted further into the brain through a complex neuronal chain.

4.2 The retina

While the outer layers of the eye serve an optical and refractionary role in the transmission of light, the retina perceives and encodes it. As such it is a highly specialized and integrated part of the central nervous system (CNS). During embryogenesis the optic cup forms as a double- layered extension of the forebrain, with which it is continuous. The inner layer of the optic cup differentiates into the neural retina (NR) centrally and into the non-pigmented layer of the ciliary epithelium (CE) and iris peripherally. The outer layer of the optic cup gives rise to three types of pigmented epithelial cells: the retinal pigmented epithelium (RPE), the pigmented CE and the iris pigmented epithelium (IPE). Thus, all of these tissues – although diverse – share a common neuroepithelial origin and form a structural and developmental continuum with the brain (Figure 1)

The important sensory pathway of the retina is damaged in common eye diseases such as degenerative retinal diseases, diabetic retinopathy, arterial occlusions, traumas and glaucoma.

There is to date no cure for such retinal damage. Once retinal injury is sustained, loss of function is permanent. However, advances in stem cell research over the last decades have sparked hopes that it may yet prove possible to restore retinal function - and ultimately vision – through stem cell based therapy. This thesis addresses the topic of adult human retinal stem cells.

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10 Figure 1. Retinal development. The neural retina, CE and IPE share a common origin. In

humans the CMZ does not persist in adulthood.

( http://faculty.washington.edu/tomreh/eyedev.html)

4.3 Stem cells, niches and the neurosphere assay

Until the 1990s it was a central dogma of neuroscience that no new neurons could be formed in the adult human brain. This doctrine was best formulated in the words of the histologist Ramon y Cajal: "Once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree." (1) In 1992 this view was challenged when it was shown that neural stem cells (NSC) with the capability to produce neurons could be isolated from certain areas of the adult brain, more specifically the subventricular zone (SVZ) (2, 3), and the dentate gyrus of the hippocampus (4, 5). Today it is accepted that neurogenesis does occur in the adult human brain, and that these new neurons are derived from NSC (6).

An adult stem cell is commonly described as a cell with the ability to both self-renew and divide asymmetrically to produce progenitor cells with a more restricted proliferation and differentiation potential (Fig. 2). A true stem cell should be multipotent, i.e. it can give rise to all cells within the tissue from which it is derived (7), while a progenitor cell is considered to be unipotent. In vivo, stem cells are thought to reside in a so called stem cell niche, where their properties are carefully regulated by the structural and functional conditions of the

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11 extracellular matrix (ECM), cell-cell interactions, and complex signalling cascades (8). One such well-characterized stem cell niche for NSC is the SVZ (9).

Fig.1. The hallmarks of a multipotent stem cell: self- renewal and proliferation to form progenitors and differentiated cells of different lineages (10).

The study of stem cells is challenging, as there are few, if any, genetic markers or morphological characteristics that precisely identifies a stem cell as such. Thus, one can only conclude that stem cells are present in a tissue sample retrospectively, based on the functional criteria of proliferation, self-renewal and production of differentiated cells (11). When studying NSCs in vitro it is common to use the neurosphere assay, first described by Reynolds et al (2), where tissue is prepared to form a single-cell suspension and cultured in a defined medium containing mitogens. After a few days free-floating clusters of cells with a characteristic rounded appearance, known as neurospheres, are formed. These were originally thought to represent stem cells and their progeny, deriving from clonal expansion of a single cell. Through repeated passaging the spheres are dissociated and replated as single cells, and the stem/progenitor cell population may be expanded. This assay, whilst a useful tool for producing large numbers of stem- and progenitor cells for further study, has also become an important method of detecting the presence of stem cells in a tissue. The continued formation of neurospheres over the course of many passages is interpreted as an expression of the stem cell’s capacity for self-renewal and the expression of mature neural and glial markers as the stem cell’s multipotentiality (11, 12).

However, the neurosphere assay has limitations. The population of cells within a sphere is heterogenous, consisting of cells at many different stages of differentiation and committed to different lineages (13-15). One must keep in mind that the sphere is not a pure stem cell population. Also, the neurosphere culturing method is sensitive to variations in factors such as

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12 cell density, concentrations of added mitogens, number of passages, etc. This can make it difficult to compare results between research groups and may account for the great variation in published results (7, 12). As described above, the properties of a stem cell is regulated by its niche. The sphere may be viewed as an in vitro niche which provides different stimuli and cues to the cells therein. Thus, depending on the inherent plasticity of the cells, they may display different potentiality in vitro than they would be capable of in vivo (12). Lastly, evidence has also been presented that non-stem cells may be capable of forming clonogenic spheres in culture (16). Since most of the evidence for the existence of retinal stem cells, as well as the results of our own publications, are based on the neurosphere assay it is important to have a clear understanding of the benefits and limitations of this culture method.

4.4 The retinal stem cell hypothesis

In adult humans, the cells of the retina and surrounding tissues (ciliary body (CB) and IPE) appear to have exited the cell cycle and to be in a quiescent state when uninjured. The retina is considered to have limited regenerative potential, and severe injuries will lead to permanent damages for which there are currently no curative treatment options.

However, in many lower vertebrates new retinal cells are produced throughout life from multipotent cells residing in the ciliary marginal zone (CMZ) (17). The anatomical location of the CMZ corresponds to the peripheral retina (PR)/CB in mammals, but while the CMZ is an immature neuroepithelial tissue, the CE of the CB consists of fully differentiated and quiescent epithelial cells arranged in a double layer (18). Considering the neuroepithelial origin of the CE and the recent discovery of NSCs in the brain, a hypothesis was formed that NSCs may reside also in the adult eye. In 2000 two research groups succeeded in isolating pigmented cells which displayed certain characteristics of stem cells from the CE of mice:

the cells proliferated clonally in culture – forming neurospheres – for several passages and expressed mature retinal genes when exposed to differentiation-promoting conditions (19, 20). Later, these results were replicated in humans (21-23).

I will here give a short outline of the evidence for the existence of retinal stem cells in the human retina at the time that this research commenced. Coles et al. attempted to culture cells isolated from the NR, pars plana and pars plicata of the CB, RPE and iris using the neurosphere assay, and found that spheres were formed only from the CB and iris. Of these, only spheres from the CB could be passaged to form secondary spheres, indicating that only

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13 cells from this location exhibited the capacity for self-renewal. Multipotentiality was inferred by the immunohistochemical detection of markers for mature retinal cells of all lineages.

Finally, cells were transplanted into developing mouse retinas, where a number of them showed signs of migrating and integrating into the host retina, as well as expressing mature retinal markers (21). Mayer et al. found sphere-forming cells in both the pars plana and the NR itself (in contrast to the study cited above). These spheres consisted of cells expressing immature neuronal and glial markers. When exposed to differentiation conditions a subset of cells expressing rhodopsin – a photoreceptor marker – were identified (22). The same group later performed a study showing that adult human retina consistently gave rise to spheres in culture irrespective of age, sex or post-mortem time (24). Xu et al characterized spheres derived from the CB, confirming earlier findings that they consist of proliferating cells that express certain immature neuronal and glial markers, while mature retinal markers could not be identified. Differentiation was not attempted (23).

Whilst the results of these studies support the adult retinal stem cell hypothesis, they have obvious weaknesses. The capability of sphere-forming ciliary epithelial cells for proliferation and self-renewal is well documented, but their multipotentiality less so. So far it has only been shown that these cells express certain mature retinal markers in culture. In order to conclude that functional retinal neurons have been formed it would be necessary to demonstrate that they are postmitotic, have the correct morphology, and are capable of firing action potentials and releasing neurotransmitters (25). Also, it is important to remember that these putative stem cells are derived from a non-neural tissue, but with neuroepithelial origin, the CE. When the current PhD project was commenced, no one had yet investigated whether the CB – derived spheres contained a pure population of neural and glial cells – like neurospheres from the brain – or if they retained part of the epithelial phenotype of the tissue from which they were derived. This would have an important impact on their status as retinal stem cells, as well on their potential use in cell-based therapy.

4.5 Strategies for stem cell-based therapy

There are various ways in which stem/progenitor cells may be put to clinical use. An important characteristic of NSCs is their ability to migrate towards sites of injury (26-29).

Thus one possibility is to achieve retinal repair through activation of endogenous cells (7).

Another option is to transplant cells into the diseased eye. The transplanted cells may exert

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14 therapeutic effects in several ways: they can be used as vectors for neuroprotective and/or disease modulating factors (30, 31), or for inhibition of pathogenic substances using short/small interfering ribonucleic acid (siRNA) technology (32). Lastly, they may replace damaged cells by functionally integrating into the host retina (21, 33).

The three main categories of stem cells which are currently being investigated in transplantation studies are embryonic stem cells, induced pluripotent stem cells (iPSC) and adult stem cells – or somatic stem cells (18). The focus of our project is on the use of adult stem cells, as these have several putative important advantages. While embryonic stem cells have a great capacity for proliferation and differentiation into all tissues of the body, their use is associated with ethical and safety concerns. Their unlimited proliferative capacity carries the unwanted risk of tumorgenicity. There is also the issue of immunological incompatibility with the host recipient (18). iPSCs are produced from somatic cells by inducing certain genes that cause their dedifferentiation into embryonic-like stem cells. This circumvents the ethical issues associated with the use of bona fide embryonic stem cells, but the danger of tumor formation and other safety issues remain, especially as these cells are genetically modified.

(34). Adult somatic stem cells have a lesser degree of proliferative capacity and potency, and as such they are considered to be safer (18, 23). The scenario of autotransplantation, where stem cells are isolated from the patient, expanded, differentiated, and/or manipulated before delivery to the site of injury, is promising, as both ethical and immunological issues may be avoided (31). The main question to be addressed regarding replacement therapy using adult stem cells, retinal stem cells in particular is whether their innate potential to proliferate, differentiate and functionally integrate is sufficient.

4.6 The IPE as a potential source of stem cells

Although studies on human tissue so far have failed to clearly demonstrate the presence of potential stem cells in the IPE (21), studies in chickens (35), rats (36), and pigs (37) have shown that the IPE harbours cells with the same stem cell – like properties as the CB. While the CB remains the best characterized potential stem cell niche in the human retina, recent studies have also presented evidence that the Müller glia (MG) of the retina display stem/progenitor properties, both in rats (38) and humans (39). Cell cultures from the human choroid, sclera, limbal epithelium and RPE have also been shown to have some potential for proliferation and neuronal differentiation (40-43).

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15 It remains to be determined which of these cell populations, if any, have inherent properties which make them suitable for cell-based treatment of retinal diseases. A separate, but related question is which source of cells within the eye would be the most practically suitable for transplantation in a clinical setting. The IPE is the only tissue in the eye of neuroepithelial origin that can be biopsied by a minimally invasive procedure, the peripheral iridectomy.

In fact there are previous reports of autotransplantation of IPE for degenerative retinal disease in humans (44-47). These efforts focused on using IPE to replace RPE, which is progressively lost in age-related macular degeneration (AMD). Importantly, it was shown that large numbers of cells – sufficient for transplantation – could be grown from single iridectomies (48, 49). Transplantation of cultured autologous IPE cells to the subretinal space in patients with AMD resulted in improved visual acuity (50), suggesting that these cells are robust enough to survive extensive manipulation in vitro, and to survive and function in vivo after transplantation to a foreign site.

Haruta et al. reported that IPE cells from rodents expressed rhodopsin after transfection with cone rod homeobox (Crx), a homeobox gene controlling photoreceptor development (51).

Later, it was shown that transfection of primate IPE cells with Crx and two other genes produced photoreceptor-like cells which expressed many mature photoreceptor markers, displayed an electrophysiological response to light, and were able to integrate into explanted retinas in vitro.(52) Again, this suggests the robustness, plasticity and potential suitability of IPE for cell-based therapy. However, these studies do not address the question of whether neuroepithelial stem-like cells reside within the IPE.

In conclusion, the IPE may be the eye’s most accessible and well-suited location for harvesting tissue for cell-based therapy. We therefore found it of importance to investigate whether the adult human iris harbors cells with properties of retinal stem cells.

4.7 Müller glia as a potential source of stem cells

Müller glial (MG) cells are the main glial cell type of the retina. Their cell bodies reside in the inner nuclear layer and their radial extensions span the entire thickness of the retina. They are descended from a common progenitor cells pool with the other retinal (neural) cell types (53).

In mammals, MG cells are quiescent, playing a protective and supportive role for retinal neurons. They contribute to homeostasis and maintain the structural integrity of the retina (54, 55). However, in certain lower vertebrates, they may also play an active role in retinal

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16 regeneration. It has long been known that the fish and amphibian retina harbors NSCs in the CMZ which contribute to retinal growth and regeneration throughout life (56, 57). These cells may also respond to injury and initiate retinal repair through generation of new neurons.

However, it was quite recently shown that retinal regeneration is also mediated by MG cells (58-60). Throughout life, they sporadically reenter the cell-cycle, proliferate and give rise to progenitor cells, which finally differentiate into functionally integrated rod photoreceptors.

This is known as the rod lineage (61). More importantly, in the case of retinal injury they may play the role of a multipotent stem cell and thus give rise to all cell types of the retina (58, 59, 62). Interestingly, this process involves only partial dedifferentiation. The Müller cell retains its structure and glial characteristics even while asymmetrically dividing to give rise to retinal progenitor cells (61). This challenges the conception of a stem cell as a more undifferentiated and immature entity.

The fact that MG have stem cell properties should perhaps not be surprising, as NSCs in the SVZ also have a glial phenotype (63). As discussed earlier, it is now an established fact that the adult human brain harbors neural stem cells. From the above discussion of MG cells and the role they play in both healthy and injured amphibian retinae it would seem that they may also be a likely candidate for adult human retinal stem cells.

In mammals, MG cells may also be activated by injury, but this activation commonly results in a pathological process known as reactive gliosis. Gliosis is a response to injury of the nervous system, mediated by activated glial cells. It may both accentuate and/or prevent further injury. Glial cells may as a result of their activation secrete neuroprotective factors and clear up cellular debris, but they may also form glial scars which disrupt tissue architecture and contribute to neurodegeneration (54, 64). Interestingly, the cellular changes involved in Müller cell gliosis are almost identical to those involved in retinal regeneration mediated by Müller cells in zebrafish. Upregulation of immature neural and glial markers such as glial fibrillary acidic protein (GFAP) and nestin is seen, and the cell cycle is reentered. However, in mammals, the proliferating cells do not differentiate into a neural direction, but rather into contractile myofibroblasts (61, 64). There is some evidence of a neurogenic response to injury in rats, where Müller cells proliferated in response to neurotoxic injury and expressed markers of amacrine cells (65) and bipolar cells and rods (66). However, this response has not been shown to be of any physiological significance.

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17 So why do mammalian MG not produce new retinal neurons in response to injury to the extent that fish and amphibian MG do? This is thought to be due to a non-permissive microenvironment/niche in the mammalian retina. When neural grafts or stem cells are transplanted to injured mammalian retinas it has been shown that survival, neural differentiation and integration is prevented by a hostile microenvironment (64, 67). One may from this infer that when Müller cells are removed from their in vivo niche and cultured in vitro they should be able to give rise to new neurons.

In 2006 it was shown that Müller cells from adult rats could be grown as neurospheres and expressed stem cell markers. Upon transplantation into mechanically injured retinae they differentiated morphologically into mature retinal cells and expressed markers of such (38). In 2009 Bhatia et al examined human cadaveric retinae for expression of stem cell markers and found a high expression of nestin in the non-laminated peripheral retina, co-staining with Müller glia-markers such as vimentin and CRALBP. Nestin-expression decreased towards the central retina, and it was not detected in the CE. Retinal explants were cultured in the presence of mitogens (epidermal growth factor; EGF), and the Müller-like cells of the peripheral retina responded with proliferation. This suggested the existence of a ciliary margin-like zone also in humans (68). Later, the same group performed a comparative study between Müller cells from the peripheral retina and ciliary epithelial cells, showing that the the Müller cells displayed far superior proliferation and neural differentiation qualities (69).

Later, human Müller cells have been shown to differentiate into rod photoreceptors (70) and retinal ganglion cells (71), and in both cases improve functional outcome after transplantation in rat models of retinal disease. These are very promising results indeed, and provide strong evidence that MG of the adult human retina are a potential source of stem cells.

4.8 The stem cell’s response to injury

The ultimate functional test of a stem cell is whether it is capable of regenerating damaged tissue. After all, the hope that the stem cells in the future will be able to heal that which previously was irreparable, is precisely the reason for the massive research efforts currently being put into this field. In order for a stem cell to exert its reparative function, it must first be able to detect and migrate towards the lesion in question. It is well known that neural stem cells in the brain have this ability (26-29). We therefore inferred that if retinal stem cells indeed exist in the adult eye they should be activated by the presence of retinal injury and respond with targeted migration towards the damaged site.

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18 Proliferative vitreoretinopathy (PVR) is a common complication to surgery for retinal detachment (RD) and after trauma. In this condition, the initial retinal injury triggers migration and proliferation of various cell types into the vitreous and retina. These form epiretinal membranes, analogous to scar tissue, which may contract and cause traction on the retina and further membrane formation in a vicious cycle, eventually lead to loss of vision.

We used PVR as a model of retinal injury and investigated whether stem cells/retinal progenitor cells in any region of the eye responded to the PVR-lesions. Such a finding would weigh heavily in favor of the retinal stem cell hypothesis.

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19 5.

Aims of the study

The overall aim of this thesis is to shed light upon the adult retinal stem cell hypothesis by characterizing different populations of potential stem cells within the adult human eye.

More specifically, we sought to

1) Compare proliferative cells isolated from the CE to neural stem cells from the adult human brain. (Paper I)

2) Investigate whether the adult human iris is a potential source of retinal stem cells. (Paper II)

3) Look for signs of retinal stem cell activation in eyes suffering retinal injury. (Paper III) 4) Determine the in vitro differentiation potential of proliferative cells from the peripheral

retina (Paper IV)

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6. Methods and methodological considerations

The research was conducted in accordance with the Declaration of Helsinki and all tissue harvesting was approved by the National Committees for Medical Research Ethics. Animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and study protocols were approved by the Animal Care Committee of the University of Debrecen, Hungary.

6.1 In vitro cultivation

6.1.1 Material for cultivation

Post mortem human eyes were enucleated from cadavers as previously described (72). The CB and the iris were carefully dissected apart. The IPE and CE were gently scraped from the underlying tissue with a cell scraper to avoid contamination with stromal cells. No attempt was made to separate the non-pigmented from the pigmented epithelial layer. The PR was carefully dissected from the corpus vitreum, to which it stayed attached after the CB was removed. A single-cell suspension was prepared by incubating the tissue in an enzyme solution, followed by careful trituration (73, 74).

Peripheral iridectomies were obtained during trabeculectomies for medical intractable glaucoma after informed written consent. Care was taken not to lose the black pigmented IPE from the rest of the iris during removal from the field of operation. The IPE was then peeled from the underlying stroma and incubated in an enzymatic solution, followed by careful trituration. The size of the iridectomies obtained during glaucoma surgery was no more than 2-3mm in diameter.

Vitreous samples were obtained after written informed consent during vitrectomies for RD with or without confirmed PVR based on evaluation of wide angle images. Cases where retinotomies, retinectomies or cutting of the retinal tear were performed were excluded from the study. The vitreous samples were centrifuged and the resulting pellets were cultivated in vitro.

Human SVZ tissue. Biopsies from the ventricular wall were harvested from temporal lobe specimens obtained during neurosurgery due to medical intractable epilepsy (75).

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21 The pigmented epithelium of the CB and ventricular wall tissue were also isolated from 3-4 week old female Brown Norwegian rats after decapitation.

6.1.2 Neurosphere culture

The primary cell suspensions from CB, iris, SVZ and vitreous samples were plated in a defined sphere-promoting medium, as we have previously described (73, 74, 76). Spheres were supplemented with growth factors twice a week, and passaged every two weeks. This involves enzymatically and mechanically dissociating the spheres to form a single-cell suspension, whereupon secondary spheres may form. Spheres were normally studied at passage 2-3, but some were kept in culture for up to six passages. Passaging the spheres has been viewed as a test of the self-renewal capacity of the cells within (11, 12). However, this is based on the assumption that the spheres are formed from the clonal expansion of a single cell. In this study we made no effort to investigate whether this was the case for our cultures.

Moreover, it has recently been shown that CB spheres, in contrast to SVZ spheres, may grow also by incorporating other spheres and adherent cells (77, 78). Therefore we can only use repeated passaging as a test of the cells’ ability to survive and proliferate in culture for extended periods of time, and not as a test of “stemness”.

6.1.3 Adherent monolayer culture

For adherent monolayer cultivation, cells were plated in medium containing 1% fetal calf serum. Monolayer cultures were split when reaching confluency. The number of cells was determined using a hemocytometer to count the number of viable cells in a 10 μl sample immediately following passaging and prior to replating. The numbers of cells were compared to sphere cultures.

We have increasingly used adherent cultivation, and in Paper IV there is no data included from spheres. This is due to the challenge involved with obtaining enough cells from neurosphere culture. Spheres derived from CB and IPE must be subjected to aggressive enzymatic dissociation in order to achieve a single-cell suspension. During this process many cells are lost or die, causing total cell numbers to drop off after repeated passaging. Adherent monolayer cultures, on the other hand, allows for expansion of cell numbers.

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6.2 In vitro differentiation

There is great variation in the differentiation protocols used by research groups studying adult retinal stem cells. This makes it challenging to compare results. It has not been determined yet which factors and nutrients are most effective in inducing differentiation in cultured cells from the CB, IPE or retina. Many protocols in use are adapted from protocols used for differentiation of NSCs from the brain, but as we and others have shown, there are important differences between putative RSCs of the adult eye and brain (16, 73), suggesting that a different strategy may be needed to optimize differentiation potential. We have in the current PhD thesis utilized several different differentiation protocols. In Paper II, spheres were dissociated to form a single-cell-suspension and plated on a poly-L-ornithine coated glass surface in a medium containing 3% FBS, B12 with addition of vitamin A and laminin. This was based on a protocol used by Tropepe et al (19). Later, Gualdoni et al published a differentiation protocol including a three day priming step and additional factors, such as Shh, taurine and DAPT. This protocol has been shown to be effective for differentiation of embryonic retinal stem cells (79), and we utilized it for differentiation of Müller-like cells from the peripheral retina in Paper IV. However, we do not know whether this is the optimal protocol, allowing the cells to differentiate to their full potential.

6.3 Immunostaining and confocal microscopy

Gene expression profiles of whole tissue samples, spheres, and differentiated cells were studied by immunohisto- and cytochemistry, using a wide range of antibodies. Methods used were peroxidase staining, fluorescent labelling of cryosections, fluorescent labelling of paraffin-embedded sections and directly fixed adherent cells.

6.3.1 Cryosections vs paraffin-embedded sections

The advantages of paraffin-embedded sections are that the tissue is better preserved, resulting in better morphology and allowing the sections to be stored for extended periods of time.

However, when using fluorescent probes, there is a larger problem with autofluorescence in paraffin-embedded sections. The paraffin-embedment-process may also mask certain antigens (80). The issue of autofluorescence can to a certain degree be circumvented by the use of confocal microscopy in the place of an epifluorescence microscope, as the confocal

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23 microscope uses light of a much narrower bandwidth and allows for analysis of very thin optical sections – both of which results in less autofluorescence (80).

6.3.2 Immunofluorescence vs peroxidase staining

In contrast to peroxidase staining, the use of fluorescent probes allows for multiple staining of several proteins in the same sample. This was of importance in our study, as we wished to search for the co-localization of several gene products. However, the presence of heavily pigmented cells in our samples caused problems with both methods. For the peroxidase staining we used a standard peroxidase technique (DAB detection kit), labelling positive cells with a brown color which was difficult to distinguish from the light-microscopic appearance of the melanin granules of pigmented cells. These granules also displayed characteristic autofluorescence under the fluorescent microscope. However, these issues were only a problem when studying very heavily pigmented samples.

6.3.3 Antibodies

Samples were labelled with antibodies targeted at a number of stem/progenitor, proliferative, neural and epithelial markers which are listed below with a short explanation.

Nestin is an intermediate filament normally expressed in neural precursors (81), and this was our main marker used to identify immature neuroepithelial cells. ATP-binding cassette transporter G2 (ABCG2) is considered a universal marker of stem cells (82). POU class 5 homeobox 1

(

Oct ¾), Nanog homeobox (Nanog) and oligodendrocyte transcription factor 2 (Olig2) are pluripotent stem cell markers, also expressed in embryonic tissue (83, 84). Paired box 6 (Pax6), SRY-box 2 (Sox2) and Cation/H+ exchanger 10 (Chx10) also known as visual system homeobox 2 (Vsx2) are transcription factors (TF)s regulating eye development (85- 87), and were used as markers of RPCs. GFAP is expressed in reactive astrocytes and Müller cells of the retina (39, 88), and was used as a marker for glial cells. Beta-III-tubulin and doublecortin (DCX) are used as young neuronal markers (40, 89). Microtubule-associated protein 2 (Map2) and neurofilament M are considered more mature neuronal markers (19, 40). Rhodopsin is a light-sensitive pigment present in mature photoreceptors (52).

Syntaxin1A (HPC1) is a docking protein found in synaptic terminals, and has been used as a marker for amacrine cells of the retina (21).

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24 Claudin is expressed in the tight junctions which connect epithelial cells (90). Cadherins are transmembrane proteins found in cell-cell adhesion structures. E-cadherin is primarily expressed in epithelial tissues (91), while N-cadherin is expressed in neuroepithelial cells (92). Connexins are components of gap junctions, which are found in a variety of cell types, including both neural and epithelial cells (91). Tenacin C and laminin are components of the basement membrane produced by epithelial cells, but can also be produced by endothelial and neural cells (93). Cytokeratin 3, 12, and 19 are cytoskeletal proteins forming intermediate filaments in epithelial cells (91), and were used as markers for such. Vimentin is also an intermediate filament, primarily found in mesenchymal cells, but expressed in a wide range of tissues. Retinoid isomerohydrolase/retinal pigment epithelium 65 (RPE65) plays an important role in the recycling of visual pigments in the retina, and is an abundant protein in retinal pigment epithelial cells (94)

Pigment epithelial-derived factor (PEDF) is a potent endogenous inhibitor of retinal angiogenesis and a neuroprotective agent normally secreted by the RPE and the CM epithelium (95-97). Ki-67 is a nuclear protein expressed at certain stages in the cell cycle (98), and is used as a marker of proliferation (99).

6.3.4 Staining procedure and evaluation

Most of the immunostaining was performed using an automated immunostaining system (LabVision Autostainer360 (Lab Vision Corporation, VT), minimizing variations in the staining protocol. Unspecific staining is a problem in all immunohistochemistry (IHC), and to minimize the effect of this we used appropriate negative and positive controls wherever possible. The sections were analyzed using an Olympus BV 61 FluoView confocal microscope (Olympus, Hamburg, Germany) and a ZEISS Axio Observer.Z1 fluorescence microscope (ZEISS, Oberkochen, Germany).

We often evaluated the expression pattern semiquantitatively, as in many cases it was difficult to precisely determine the number of positive cells in a sample. Some cells were clearly positive, others clearly negative, but there were also a number of cells that were indeterminate. Two independent investigators were used to evaluate the expression pattern.

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6.4 Ultrastructural analysis

In order to more comprehensively characterize the cells, we used electron microscopy to study their intracellular structure and morphology, as this provides important phenotypic information to supplement the analysis of gene expression. Transmission electron microscopy (TEM) was used to look for specific neural and epithelial structures within the spheres, such as specialized cell-cell junctions and pigment granules. Scanning electron microscopy (SEM) was used to assess the spheres’ surface morphology.

6.4.1 TEM

The spheres were fixed for 30-60 min at room temperature by immersion in freshly prepared mixed aldehyde-fixation containing 0.1M sodium cacodylate buffer, 2% glutaraldehyde, 2%

paraformaldehyde and 0.025% CaCl2, pH 7.4 (100). Fixation was continued overnight at 4 °C, postfixed in 1 % osmium tetroxide, and dehydrated through a graded series of ethanol up to 100%. The spheres were then immersed in propylene oxide for 20 min and embedded in Epon (Electron Microscopy Sciences, Hatfield, PA). Ultra-thin sections (60-70 nm thick) were cut on a Leica Ultracut Ultramicrotome UCT (Leica, Wetzlar, Germany) and examined using a CM120 transmission electron microscope (Phillips, Amsterdam, the Netherlands).

6.4.2 SEM

Mixed aldehyde-fixated spheres were dehydrated in increasing ethanol concentrations, packed in filter paper and dried according to the critical point method (Polaron E3100 Critical Point Dryer, Polaron Ecq. Ltd, Watford, UK) using CO₂as the transitional fluid. The spheres were gently transferred to carbon stubs and coated with a 30nm thick layer of platinum in a Polaron E5100 sputter coater (Polaron) before being examined and photographed with an XL30 ESEM electron microscope (Philips, Eindhoven, the Netherlands).

6.5 Gene expression

6.5.1 Real-time quantitative reverse transcription PCR (qRT-PCR)

qRT-PCR is a four step procedure: 1.RNA isolation, 2.RT cDNA synthesis, 3.PCR and 4.detection and quantification of PCR products reflecting RNA levels and calculate differences in RNA levels and gene expression between samples. This technique was

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26 employed in order to provide further information on gene expression patterns. Unspecific staining may be a problem in IHC, so where possible we sought to confirm the presence of gene products both at the messenger ribonucleic acid (mRNA) and protein level. Moreover, we were able to study a larger number of genes with PCR, and the use of qPCR allowed us to more precisely quantify the up- or downregulation of genes than the semiquantification method applied for immunostaining. However, this method also has inherent limitations. An increase or decrease in protein levels are not always reflected by changes in mRNA levels regulated by transcription (synthesis of RNA from a DNA template). Protein levels are also regulated by the degradation time/turnover time of mRNA molecules and by factors affecting translation (synthesis of the protein from the mRNA template). This can explain the discrepancies sometimes found between immunostaining and qRT-PCR.

Good quality RNA is essential to reflect the real time gene expression by qRT-PCR. The RNA quality and integrity was evaluated by Agilent 2100 BioAnalyzer (Agilent technologies, Santa Clara, CA, USA). RNA quantity and purity was evaluated by spectrophotometry (Nanodrop, Wilmington, DE, USA). RNA impurity could affect the quantification of RNA, so there is less or more input in the cDNA synthesis and the PCR reaction than intended. RNA impurity could also affect cDNA synthesis and PCR efficiency, further causing fold-change differences in gene expression, not reflecting the RNA levels in the samples.

TaqMan technology was used for the qRT-PCR. We also use pre-designed TaqMan gene expression assays (Life Technologies) using primer/probes that have been designed and tested to be gene-specific and have a high PCR efficiency. TaqMan, although expensive, has several advantages compared to e.g. SYBRgreen. It has a higher specificity and is less laborious.

There is no need to check primer specificity, PCR efficiency or running melting curves to verify that there is only one PCR product and no primer-dimers. We use “master mixes” for the qRT-PCR to have as little variety as possible in the mixes, thus as little as possible variety in the PCR efficiency between the samples and the runs. We also use “no template control”

for each “primer mix” to be sure that the amplification seen is not due to cross-contamination by any other template. It is important to also use “minus RT control”: cDNA synthesis without adding the RT enzyme. Then if there is any product amplified in the PCR, this is an indication of contaminating genomic DNA also present in and affecting the other qRT-PCR reactions.

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27 It is important to select a reference gene that is equally expressed in all samples as an endogenous control, so it reflects the levels of material (input) used for each sample and could be used to adjust the gene expression accordingly. 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin are often used as reference or input control genes. The optimal reference gene, equally expressed in all samples, depends on the cell types included in the study, and this must always be tested. We used 18S rRNA in Paper 1, and thereafter GAPDH, as we found that this gene had the most stable expression across the samples.

For analysis of the data acquired, we used the StepOnePlus Real Time PCR system or Sequence Detector Software (version 1.6.3, Applied Biosystems). Data was analyzed by the comparative Ct method (2^-ΔΔCt). The threshold cycle (Ct) is the PCR cycle in which the fluorescent signal of the dye reaches a specific threshold. The Ct is inversely related to the amount of PCR product, i.e., the lower the Ct value, the greater the number of PCR products and the greater the number of RNA molecules in the initial sample. Several methods have been used to calculate and present relative gene expression, of which the most widely used method, is the comparative Ct method (101). In Paper II the PCR products were visualized using the MCE-202 MultiNA Microchip Electrophoresis System for DNA/RNA analysis.

6.5.2 Microarray

The microarray is a method for determining the expression pattern of a large number of genes simultaneously. A microchip marked with multiple olignucleotide probes, complementary to known genes, is used for this analysis. As for the qRT-PCR, RNA must first be isolated and assessed for purity and quality. cDNA synthesis is performed, the cDNA is labelled and then applied to the microchip, where the sequences of interest are detected through hybridization to the probes on the chip and analysis of the fluorescent signal.

This technique has the advantage of detecting new genes of interest, as the number of genes studied is much higher than what is practically feasible with RT-PCR. However, sensitivity is lower, and results depend on multiple factors, such as quality of labelling, hybridization efficiency and changes in the fluorescent signal. This may explain some of the discrepancies between microarray data and qRT-PCR data found in our study (Paper IV).

Microarray analysis was performed on cells from three donors before and after 7 days of differentiation, using the Affymetrix GeneChip Human Gene 2.0 ST which contains about 40,000 gene transcripts. Signal intensities were detected by Hewlett Packard Gene Array

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28 Scanner 3000 7G and Robust microarray analysis (RMA) was applied for normalization and generation of signal values. Gene transcripts with maximal signal values of less than 32 across all arrays were removed to filter for low and non-expressed genes, reducing the number of gene transcripts to 25 644. For expression comparisons of experimental groups, a two-way ANOVA model was used. The resultswere expressed as fold change (FC) with p-values.

Regulated genes, FC>2 and p<0.05, from the gene expression analyses were uploaded into the Ingenuity Pathways Analysis for organization into functional categories. Microarray analyses were performed by a skilled researcher at the core facility at Oslo University Hospital, Ullevål.

6.6 Mouse model of proliferative vitreoretinopathy induced by dispase

In order to reproduce the pathological environment of PVR formation in a controlled animal study, we utilized a mouse model of PVR induced by intravitreal injection of the proteolytic enzyme dispase. This model is known to induce glial activation as well as both epi – and subretinal membrane formation (102, 103). Animal models offer the advantage of standardized conditions, with less variation among samples and less confounding factors.

However, there may be important species differences to be considered. Results should always be verified using human tissue if possible. Such species differences may account for the discrepancy in results between mouse and human eyes in Paper III. It is of great interest to document such differences, as much of the research on putative RSCs in the adult eye has been performed in rodents.

Female 4-6 months old wildtype mice were anestesized and received intravitreal injections with 4µ dispase, as previously described (102). Control animals received 4 µl of sterile physiological saline solution. Stratus Optical Coherence Tomography images (OCT) were taken following injections to monitor disease progression. Control and dispase treated mice were sacrificed between 7 and 14 days following injections when signs of PVR formation were evident. PVR formation was validated in cryosections by the presence of cellular hyperplasia, retinal folding, and GFAP⁺ epiretinal membranes.

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7. Summary of results

7.1 Paper I

Previous studies have shown that proliferative cells isolated from the adult human CB display properties of NSCs (21, 23). This study directly compares the expression pattern of neural and epithelial markers, as well as morphological characteristics, between adult human CB – and brain – derived spheres.

We found that the ciliary epithelial cells proliferated to form pigmented spheres which grew significantly faster than the non-pigmented spheres from the SVZ. The CB spheres, in contrast to the SVZ spheres, expressed cytokeratins, a marker of epithelial cells. They also expressed significantly lower levels of neural progenitor markers, such as nestin, Sox-2 and GFAP, as shown both by immunostaining and qRT-PCR. Interestingly, even the nestin – positive cells in the CB spheres contained pigment granules.

SEM of the spheres showed different surface patterns. The CB spheres displayed a smooth surface of flattened cells, while the surface of the SVZ spheres was covered in spherical cells.

TEM showed that both CB and SVZ spheres contained a mixed population of cells embedded in ECM. In the periphery of CB spheres we found elongated and polarized epithelial – like cells which were connected with desmosome – and tight junction – like structures. Although some polarized cells were also found in the periphery of SVZ spheres, there were no signs of desmosome – and tight junction – like structures.

qRT-PCR also showed significant differences in the expression of genes related to growth factor signaling. There was higher expression of EGF, transforming growth factor- beta (TGF-β) and bone morphogenetic protein (BMP) receptors in the CB spheres and a comparatively greater activation of the canonical Wnt pathway, while there was a lower expression of fibroblast growth factor (FGF) receptor type 2. Immunostaining showed that CB spheres, in contrast to the SVZ spheres, produced PEDF, which is a potent endogenous inhibitor of retinal angiogenesis and a neuroprotective agent normally secreted by the RPE and CE (95-97)

In conclusion, the CB spheres contain cells with epithelial properties and limited expression of neural progenitor markers compared to SVZ neurospheres.

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7.2 Paper II

We further wanted to investigate whether the adult human iris may be a source of retinal stem cells.

IPE cells from peripheral iridectomies and human post mortem tissue proliferated in culture to form pigmented spheres which could be passaged repeatedly, partially losing their pigmented appearance. We used RT-PCR and immunostaining to compare whole tissue, primary spheres and tertiary spheres. CB spheres were used as a control.

We found that the IPE and CB cells both expressed a variety of both neural and epithelial intermediate filaments as well as several eye field TFs expressed in retinal stem/progenitor cells. These expression patterns were mostly conserved through extensive in vitro culture, both using the neurosphere assay and a minimal serum adherent monolayer culture system.

We found that the spheres contained a distinct population of nestin+ cells which showed no double staining with epithelial markers. Both the nestin+ and nestin- cells were proliferating.

Ultrastructural characterization of IPE spheres revealed a similar structure to that of the CB spheres: they contained a mixed population of cells embedded in ECM, connected to each other partly with gap- like and adherence- like junctions (such as are found in neurospheres from the SVZ), but also with desmosome- and tight- like junctions, which are found in CB spheres and not in SVZ spheres.

A minimal serum adherent cultivation protocol was used for expanding IPE cells. This greatly increased the number of cells compared to the neurosphere assay, while the cells were not immortalized, nor did they lose their neuroepithelial properties.

When exposed to differentiation-promoting conditions cells migrated out of spheres and a few of them developed a mature neuronal- like morphology with a polarized appearance and long dendrites, as well as cells with round cell soma and short extensions. These cells expressed mature pan-neuronal markers, and some co-staining with rhodopsin, a marker of photoreceptors, was observed. However, we did not detect markers of other mature retinal neurons or glial cells.

In conclusion, proliferative cells with limited retinal stem cell properties can be isolated from human peripheral iridectomies.

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7.3 Paper III

Having determined that both the CB and iris of the adult human eye harbours cells with certain characteristics of retinal stem cells, the next step was to determine whether these cells are activated in response to retinal injury.

We first mapped the expression of neural and retinal stem cell markers in normal eyes. Nestin was expressed in a few cells in the central retina, but most strongly in walls of cysts in the peripheral retina and most proximal pars plana, where cells positive for Pax6 and Sox2 were also found. No nestin+ cells were found in the peripheral pars plana or pars plicata of the CE, nor in the IPE.

In eyes with PVR the nestin staining extended further into the proximal pars plana, and two populations of cells were identified: one Nestin+/GFAP+ and one Nestin+/GFAP-. There was evidence of active proliferation, indicated by nuclear Ki67-staining. Although we detected an increase in cell division also more peripherally in the CE, no markers of NSCs and no rhodopsin+ cells were found in the peripheral pars plana, pars plicata or IPE.

We then utilized a mouse model for PVR. In contrary to the findings in humans, scattered nestin+ cells were found in the CE of control eyes, and nestin expression was upregulated in the CE of PVR eyes.

Vitreous samples from patients undergoing vitrectomy for RD were studied, and we could isolate sphere-like structures from them. Most of the cells within these structures stained for both nestin and GFAP. Cells isolated from such samples were grown in culture to assess their sphere-forming capacity. We found that spheres formed more often from patients with confirmed PVR, that these could be repetitively passaged and that they expressed nestin, GFAP and beta-III-tubulin.

Finally the gene expression profile of PVR-derived spheres, CE spheres and cultures of retinal cells were compared using qRT-PCR. Importantly, GFAP was found to be much more highly expressed in PVR spheres compared to CE spheres. Also, the expression of nestin in all groups was comparable, even though we did not detect nestin expression in the CE in situ.

In conclusion, the adult eye does harbour cells with certain stem cell qualities that are able to respond to retinal injury, but these appear to be cells with glial characteristics located in the PR – and not in the CB or iris.

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7.4 Paper IV

After concluding that cells derived from the adult human CB and iris only have limited stem cell characteristics and do not respond to retinal injury in vivo, we proceeded to look more closely at the population of retinal cells which were activated by injury in Paper III, namely the glial-like cells of the PR. The goal of this study was to characterize in vitro differentiation potential of the glial-like cells of the PR.

We found that the unlaminated PR contained a high density of cells co-staining for Nestin and GFAP. The laminated central retina contained only a few such cells, and the CE contained none.

In vitro, cells isolated from the PR proliferated and expressed nestin, Pax6 and GFAP. They were then cultured under conditions known to induce differentiation for 1-5 weeks. After 1 week there were still some proliferating nestin+ cells, but there were three other distinct cell populations: cells with neuronal morphology staining for beta-III-tubulin, cells with fusiform glial morphology staining for GFAP and small rounded cells staining for rhodopsin. qRT- PCR showed a significant upregulation of the photoreceptor markers: neural retina leucine zipper (NRL), CRX, rhodopsin (RHO) and recoverin (RCVRN), the Müller and RPE-marker retinaldehyde binding protein 1 (RLBP1/CRALBP), the early RPE marker melanogenesis associated transcription factor (MITF), and the immature glial marker GFAP.

From week 2 and onwards the expression of all these markers declined markedly.

Immunocytochemistry at week 3 still showed some GFAP and rhodopsin+ cells, both of which had developed longer projections. Some cells were still nestin+.

Microarray analysis was performed at day 7, which was the time point with the greatest upregulation of differentiation markers. The number of transcripts differentially regulated when p<0.05 and FC>2 was 338 (upregulated) and 203 (downregulated). 23 of the regulated transcripts have been shown to be involved in eye development, however, some of these were upregulated, and others downregulated. It was thus difficult to draw any clear conclusion from the microarray analysis as to the multipotency of the studied cells.

In conclusion, proliferative cells isolated from the PR transiently upregulated markers associated with several retinal lineages upon induced differentiation. Stable and convincing differentiation was not seen.

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8. Discussion

I will here perform a reevaluation of the retinal stem cell hypothesis, based on the findings in our studies. I will also discuss the potential clinical implications of our results.

8.1 Does the adult human CB harbor stem cells?

In order to answer this question we first examined how the morphological characteristics and gene expression profiles of sphere-forming cells of the CB compared to those of brain-derived NSCs (Paper I). We found that CB spheres contained a population of proliferative epithelial- like cells with decreased expression of NSC markers compared to CNS neurospheres (73).

These results are partially in agreement with a recent study by Cicero et al., showing that although cells of the CB are able to clonally proliferate to form spheres and express certain markers of retinal stem/progenitor cells in culture, each cell still contained pigment and displayed membrane interdigitations and epithelial junctions, characteristics of a differentiated ciliary epithelial phenotype (16). Another recent study demonstrated that although CE cells in culture expressed significant levels of pluripotent and retinal progenitor markers, they consistently failed to differentiate into photoreceptors (104). A study that separated the pigmented and non-pigmented CE found that only the non-pigmented CE proliferated to form spheres in culture, expressing high levels of epithelial markers, very limited numbers and levels of neural progenitor markers, and could not be induced to show signs of proper neural differentiation (69)

In light of this recent evidence, it is necessary to reevaluate the retinal stem cell hypothesis.

As described in the introduction, a stem cell must have the ability to a) self-renew, b) divide to form progenitor cells and c) give rise to functional terminally differentiated cells. These stem-cell-functions are characteristically triggered by tissue injury. The self-renewal and proliferative capacity of CE cells is well documented (19, 21, 23, 73). There is also little doubt that CB spheres contain a population of cells that display certain characteristics of neuroepithelial progenitors. We have shown that CB spheres do express a range of immature neural and retinal markers (Paper I-III). Importantly, we found that the spheres contain two distinct populations of cells: one nestin+ and one claudin-1+, with no cells displaying double- staining (Paper II). This suggests that in contrary to the conclusion drawn by Cicero et al. that CB spheres consist of a homogenous population of ciliary epithelial cells, they contain both epithelial cells and cells with a larger degree of neural competence.

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34 However, expression of certain progenitor markers in vitro is not sufficient evidence of the presence of true stem cells. Kohno et al. showed that CB spheres initially consist of nestin- negative epithelial-like cells that begin to express nestin during cultivation. These spheres had the ability to grow non-proliferatively by incorporating adherent nestin- cells, which then became nestin+ (78). It was later shown that CE cells rapidly upregulate nestin during the first 24hrs in culture, before they have time to clonally proliferate (16). Thus it is possible that the cell population in CB spheres with neuroepithelial properties is not derived from true NSCs residing within the CB, but rather from a trans/dedifferentiation process where ciliary epithelial cells respond to stem cell culture conditions by shifting their gene expression profile in an immature direction.

In order to shed further light on this, we performed RT-PCR (Paper II) and immunostaining (Paper III) on wild-type CB tissue, and compared the expression of neural and epithelial genes to that of CB spheres. Immunostaining showed that the only nestin+ cells were found in the most central part of the pars plana and PR, and not at all in the rest of the CB. Thus, the expression of nestin in CB-spheres may be ascribed to contamination by cells from the PR during the tissue dissection process as well as by the influence of culture conditions.

The final test of a stem cell is in its capacity for producing differentiated cells. In this thesis, we have only assessed the differentiation potential of IPE and MG cells, which I will discuss later. Some research groups have shown that CE cells can be induced to express markers of mature retinal neurons (19-21, 37, 105). However, others have more recently shown that when exposed to differentiation conditions the CE cells tend to revert to the differentiated state of ciliary epithelial cells, and not of retinal cells (16, 69, 104). This lack of consistence in results could be caused by differences in culture protocols, but could also be due to the fact that only the latter studies have looked for morphological and genetic characteristics of epithelial cells, while the earlier ones exclusively focused on neural and retinal markers. In order to reach a final conclusion on this topic it would be necessary to perform functional studies to show that CE cells not only are capable of upregulating certain mature retinal markers in vitro, but possess the intracellular structures necessary to respond appropriately to light, form synapses, and fire action potentials. To my knowledge, this has not been done previously and would be an interesting topic for further research.

One final way of assessing the stem cell-potential of ciliary epithelial cells is to examine their response to retinal injury. We hypothesized that if retinal stem cells indeed reside within the

In vitro characterization of adult human retinal stem cells