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Okinawa Institute of Science and Technology (OIST) Workshop



‘Keeping an eye on Neural Stem Cells in light of Photoreceptor Degeneration’

Carla B Mellough

Okinawa Institute of Science and Technology (OIST) Workshop
‘Retina: Neural Stem Cells and Photoreceptor Degeneration’

Intensive research efforts worldwide have now brought us closer to understanding how we might drive the differentiation of stem cells towards functional somatic phenotypes and improve cellular integration for visual reconstitution in various forms of retinal degeneration. The therapeutic implications of this work are clear. The recent Retinal OIST International Workshop in Okinawa, Japan, organised by Ichiro Masai (OIST) and Sumiko Watanabe (University of Tokyo), brought together many of the world leaders in the development of stem cell therapy for retinal degeneration to discuss recent progress in the field.

Once the photoreceptors of the mammalian retina are lost they are not spontaneously regenerated. Various ocular and extra-ocular cell types are being investigated as a source of new neurons for the treatment of retinal degeneration. For example, research has shown that a subset of Müller glia have the potential to de-differentiate then generate amacrine cells in the mouse retina (Karl et al., 2008), the expression of photoreceptor antigens in adult iris tissue can be achieved with gene transfer (Haruta et al., 2001) and neural-derived cells can migrate well into the retina and develop morphology similar to retinal cell types (Harvey et al., 2006; West et al., 2009). However, the development of these cell types towards fully mature functional retinal phenotypes following transplantation remains unresolved. In the best case, postmitotic mouse embryonic photoreceptor precursors expressing the neural retinal leucine zipper (Nrl) transcription factor grafted subretinally into a mouse model of retinal degeneration integrated within the outer nuclear layer (ONL) and elicited some improvement in the visual response, a result that was not achieved following transplantation of proliferating or stem cells (MacLaren et al., 2006). This study provides vital information about the optimal stage of donor cell differentiation for improved survival and engraftment within host tissue, however the lack of equivalent donor material for human therapy means that another tissue source is required. It is for this reason that many researchers are turning to human embryonic stem cells (hESC) lines and patient-derived induced pluripotent stem cells (iPSC) for the development of cell therapy.

In the beautiful surrounds of Okinawa, Japan, the theme of the OIST workshop was neural stem cells and photoreceptor degeneration. The content of presentations ranged from ‘hardcore’ retinal development and cell fate specification to the applicability of stem cell therapy following photoreceptor loss and improved transplantation methods. Here, many of the highlights are presented.

Thomas Reh (University of Washington, Seattle) opened the first session by discussing the applicability of cell based therapies following photoreceptor loss. Intrinsic retinal stem cells were first introduced and then research in the chick was discussed which showed that, after N-methyl-D aspartic acid treatment, Müller glia switch on proliferation-associated genes and subsequently neural genes and can differentiate into neurons, amacrine, bipolar and ganglion cells, with similar results shown by others in rat (Ooto et al., 2004) and mouse (Karl et al., 2008). After light damage in the mouse a small subset of Müller glia can generate amacrine cells even though light damage affects the photoreceptor cells and not amacrines. However, no photoreceptor replacement was detected from Müller glia cells leading this author to turn their attention to another stem cell source, embryonic stem cells. Subsequent discussion dealt with how human ESC can be efficiently differentiated towards a photoreceptor phenotype by promotion of anterior neural fate via antagonism of BMP and Wnt/β-catenin signalling pathways in the presence of IGF-1 then FGF (Lamba et al., 2006). It was further shown that following subretinal engraftment into adult Crx(-/-) mice (a model of Leber's Congenital Amaurosis) these hESC-derived photoreceptor precursors could restore some of the light response (Lamba et al., 2009).

The role of various signalling pathways in retinal progenitor proliferation and neurogenesis featured throughout many of the presentations. Brian Link (Medical College of Wisconsin) discussed how cell polarity and membrane dynamics cooperate to regulate retinal neurogenesis. He demonstrated how proliferating cells change their position in the neuroepithelium from apical to basal and back depending on the phase of the cell cycle they are in, with cell division occurring at the apical surface and basally positioned cells more biased to form neurons. The importance of actomyosin activity during the G2 phase of the cell cycle was stressed in addition to the polarised signals of the neuroepithelium and differing transcriptional signals within apically located nuclei (Baye & Link, 2007). The role of hedgehog (Hh) signalling and the sonic hedgehog (Shh) antagonist Suppressor Fused (Sufu) in the regulation of retinal progenitor cell (RPC) proliferation and cell fate was presented by Valerie Wallace (Ottowa Health Research Institute, Canada) who demonstrated the transient overgrowth of ventral RPCs in acute Sufu knockouts, characterised by depression of Hh target genes and disruption of the RPC gene expression signature. Valerie described how the resulting dystaxic ventral retina comprises tyrosine-hydroxylase positive amacrine/horizontal-like cells, indicating multilineage competence is lost, and that this effect is Gli2-dependent; if Gli2 is also knocked out, then the normal phenotype is regained (Wall et al., 2009). This work emphasises the importance of Hh signalling for RPC proliferation in the mouse.

The role of the notch signalling effector Hairy1 was shown to act downstream of Wnt2b to maintain ciliary margin zone progenitor cells in the chick (Fumi Kubo, RIKEN, Japan), whilst the importance of sumoylation activity for Xenopus RPC proliferation (Koji Terada, Osaka Bioscience Institute) and mammalian photoreceptor development (Seth Blackshaw, John Hopkins, USA) was also highlighted. Jin Woo Kim (Korea Institute of Science and Technology) described the action of the phosphatase tensin homolog (Pten) in regulating PI3K-Akt signalling to Notch. This work shows that mouse RPCs lacking Pten complete neurogenesis earlier and show precocious depletion of the RPC population. Using advanced labelling techniques, Harukazu Nakamura (Tohoku University) revealed evidence for a new retinotectal projection pathway near the dorsal margin of the tectum in the chick, the origin, endpoint and function of which are now being determined.

Kunimasa Ohta (Kumamoto University, Japan) described the progression of his research from addressing basic developmental biology questions such as why all ganglion cell axons exit the eye at the optic nerve (Ohta et al., 1999) and the role of the lens in normal retinal formation (Ogina & Yasuda 2000). Further research into the role of Tsukushi (so named as its pattern of expression in the chick primitive streak looks like the Japanese horsetail plant of the same name) as a novel Wnt signalling inhibitor was also discussed, which affects the RPCs of the ciliary body epithelium cells, the iris pigmented epithelium cells and Müller glia and may act to maintain the growth and undifferentiated state of stem cells (Ohta et al., 2008).

Sumiko Watanabe (University of Tokyo, Japan) presented her impressive and intensive research efforts which work towards the detailed characterisation of the cell lineages present during mouse retinal development, by screening the retina with 137 commercially available antibodies. This work demonstrates, for example, that SSEA-1 (CD15) and c-kit (CD117) represent the RPC population, whilst CD26 marks the retinal pigmented epithelium and CD73 the photoreceptor precursor cells. These results provide excellent information on how one can use cell surface markers to ascertain the spatial and temporal transitions that occur in the developing retina, and the signalling pathways that act on subsets of cells at defined developmental stages (Koso et al., 2009).

Much new knowledge relating to photoreceptor development and differentiation also featured in the workshop. Otx2 is a transcription factor understood to be involved in photoreceptor cell fate determination. Additional roles for the Otx2 transcription factor in ribbon synapse and primary cilia formation was discussed by Takahashi Furukawa (Osaka Bioscience Institute) who presented the results from microarray analysis of the Otx2 conditional knockout mouse. Kwang-Wook Choi (KAIST, Korea) described the utilisation of genetic tools to discern polarity gene functions in the eye and presented work on the protein complexes involved in epithelial cell polarity and the apical basal organisation of Drosophila photoreceptors. Chromatin organisation is thought play a crucial role in controlling gene expression during development. Itsuki Ajioka (Keio University School of Medicine, Tokyo) discussed the important role of the retinoblastoma tumor suppressor protein (pRB) in rod gene expression and histone modifications during chromatin condensation. Indeed, pRb is crucial for rod maturation and global heterochromatin formation and a rod specific epigenetic modifier, Jmjd2c, was revealed. The development of more efficient methods by which to derive photoreceptor enriched cell populations would potentially provide an unlimited supply of de novo photoreceptors for transplantation into patients affected by photoreceptor degeneration. Carla Mellough (Centro de Investigación Príncipe Felipe, Spain) presented work which shows that using a specially adapted differentiation protocol, highly enriched populations of cells committed to a photoreceptor fate can be efficiently derived from hESC over a 60 day period. Douglas Campbell (RIKEN, Japan) presented work which has investigated caspase 3 mRNA transport along retinal ganglion cell axons and which reveals a role for caspase 3 protein in arborisation and synaptogenesis. Using time lapse imaging, the effects of caspase 3 inhibition on growth cone navigation, arborisation and presynaptic puncta were demonstrated. These results indicate that caspase 3 acts downstream of arbor-regulating ligand receptor systems and acts to prevent premature arbor maturation.

Robin Ali, Anand Swaroop, and Masayo Takahashi gave impressive presentations which covered some of their intensive work to date in the field and provided an excellent overview of the progress we have made thus far. Robin Ali expanded on some of his recent work which attempts to overcome one of the physical barriers to cell integration, the outer limiting membrane (OLM). The OLM is formed by adherens junctions between Müller glial end feet and photoreceptors and is a natural barrier between the subretinal space (the usual site of cell injection for photoreceptor replacement) and the photoreceptor layer. It was explained that cell integration is much higher in a mouse model of progressive OLM disruption, but that the OLM remains intact in many forms of retinal degeneration. A technique which uses small interfering RNA directed against ZO-1 (an OLM adherens junction associated protein) to induce the targeted, transient disruption of the OLM was presented. When combined with subretinal transplantation, Robin demonstrated that cellular integration was significantly improved (West et al., 2008). Anald Swaroop expanded on the transplantation issues and emphasised that successful integration of photoreceptors was heavily dependent on the age of the host, the developmental stage of transplanted cells and the time from injury to transplantation. Masayo Takahashi discussed the implications of patient-derived iPSCs on retinal regenerative medicine and using optical coherence tomography images showed examples of her observations on retinal degeneration in the human and the importance of a healthy retinal pigment epithelium if photoreceptor replacement is to be successful.

Müller glia have a wide range of functions in the retina including supply of the end products of anaerobic metabolism, clearance of waste products and neurotransmitters, phagocytosis of debris, synthesis of retinoic acid, the control of retinal homeostasis and also provide a support structure. Further to the Müller glia story presented by Robin Ali, Jimmy de Melo (Johns Hopkins University) presented work on the LIM homeodomain transcription factor, Lhx2. With its role in the retina previously undetermined, Jimmy demonstrated the selective expression of Lhx2 in RPCs and in developing and mature Müller glia and its role in regulating RPC proliferation and Müller glia terminal differentiation. Further, it was shown that loss of Lhx2 function in adult Müller cells results in Müller cell gliosis, but that this does not affect p27kip1, unlike other genetic models of reactive gliosis.

New insights into photoreceptor degeneration were provided by Ching-Hwa Sung (Cornell University, New York) who discussed the structural role of rhodopsin in photoreceptor outer segment morphogenesis and showed that disturbance of the vesicular delivery of rhodopsin in the outer segment leads to aberrant vesicle formation akin to that which is observed in various models of retinal degeneration. The mechanism of cone photoreceptor remodelling in the retinal degeneration (rd) mouse, a model of human retinitis pigmentosa, was explained by Bin Lin (Harvard Medical School, Boston). Degeneration of rod photoreceptors in this model of retinal degeneration is almost complete by 30 days after birth, however it was shown that cone photoreceptors in this model maintain a relatively normal phenotype during the period of extensive rod photoreceptor loss. In humans, the secondary degeneration of cone photoreceptors is much more debilitating than the initial phase of rod loss, as cones allow acute vision under photopic conditions whereas rods function optimally under dark conditions. It was suggested that this may allow a longer therapeutic window for photoreceptor replacement in the human than was originally thought. The neuroprotective role of bone marrow (BM) derived microglia in retinal degeneration was presented by Atsushi Otani (Kyoto University) who demonstrated that in chimeric mice with GFP-positive BM, BM-derived cells are recruited into the degenerate retina and differentiate into microglia in close apposition to degenerating vessels and neurons. Inhibition of BM-derived cell recruitment or systemic depletion of myeloid progenitors accelerated the degenerative process whilst stimulation of myeloid lineages decelerated retinal degeneration, especially promoting cone photoreceptor survival, implicating a protective role for BM-derived microglia in this inherited model of retinal degeneration.

Implications for the translation aspects of vision research were presented by Noriyuki Azuma (National Centre for Child Health and Development, Tokyo), Zi-Bing Jin (RIKEN, Japan) and Florian Gekeler (University of Tϋbingen, Germany). Noriyuki Azuma described novel Pax6 mutations in a patient with foveal hypoplasia and novel mutations of the Shh gene in two patients with holoprosencephaly and dislocated fovea. Zi-Bing Jin described the generation of iPSC lines from 7 patients with retinitis pigmentosa. Using immunocytochemical techniques and gene expression profiling the multipotentiality of these patient-derived iPSC was demonstrated and evidence for their differentiation towards retinal pigment epithelium, RPC and photoreceptor precursors following defined in vitro differentiation was presented. A particularly impressive piece of translational research was presented which lended itself perhaps more to restorative technology rather than stem cell biology, but which yielded some inspiring results. Florian Gekeler demonstrated the successful transplantation of subretinal multielectrode implants in blind retinitis pigmentosa patients. These act by electrically stimulating the remaining inner retinal circuitry following photoreceptor depletion. Using video footage, the implantation procedure was demonstrated and then the resulting restoration of letter reading, stripe pattern recognition and object recognition in one patient. The presence of the multielectrode implant generated retinotopically correct patterns which allowed this level of visual restoration to occur, even under low luminance conditions; a very impressive result. Watching this patient ´look at´ his hand and describe what he could see after being blind was particularly moving.

It is clear that we are making great advances in our understanding of retinal development, biology, pathophysiology and the treatment of retinal disease. Much improved methods in which to drive photoreceptor differentiation from hESC have emerged and the optimisation of culture conditions, cellular selection and surgical techniques can yield much improved engraftment of donor cells. But what hurdles remain before such expertise can be successfully applied clinically? We must be cautious that we fully understand the biology underpinning our observations before such therapy can be safely and successfully transferred to the clinic. For example, grafted cells do not solely integrate into the ONL following photoreceptor loss; Thomas Reh described some inner retinal integration of subretinally grafted donor cells in degenerative mutants, a common observation in publications of this nature, and it remains unclear the effect that these abberantly integrated cells will have on retinal function in human patients. Tumorigenicity is also a concern when using hESC and must be tested for stringently prior to any translation. Selection for postmitotic or specific lineage marker expressing cells may alleviate this concern, although Masayo Takahashi in her presentation suggested that 100% purification is difficult. Furthermore, as mentioned by Robin Ali, even if we facilitate the entry of cells into the retina from the subretinal space, the level of integration is still lower in at later stages of retinal degeneration. There are many questions which remain to be answered and the optimisation of protocols is ongoing. Nonetheless, we all left the beautiful Island of Okinawa with a feeling of great optimism that the small advances we are making bring us ever closer to understanding how we might work towards restoring visual function in patients affected by retinal disease and alleviate the burden of blindness.

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