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SummaryStem cell therapy for retinal disease is under way, and severalclinical trials are currently recruiting. These trials use humanembryonic, foetal and umbilical cord tissue-derived stem cellsand bone marrow-derived stem cells to treat visual disorderssuch as age-related macular degeneration, Stargardt’s diseaseand retinitis pigmentosa. Over a decade of analysing thedevelopmental cues involved in retinal generation and stem cellbiology, coupled with extensive surgical research, have yieldeddiffering cellular approaches to tackle these retinopathies. Here,we review these various stem cell-based approaches for treatingretinal diseases and discuss future directions and challenges forthe field.

Key words: A ge-related macular degeneration, Retinitispigmentosa, Stargardt’s disease, Stem cells, Clinical trials

IntroductionAlthough the retina is a complex structure, many diseases of theouter retina can be attributed to the degeneration of a relativelysimple epithelial monolayer: the retinal pigment epithelium (RPE).Although the RPE is not a defined part of the retina, it is anessential supporting tissue involved in retinol cycling, nutrienttransport, growth factor production and phagocytosis of the fragilephotoreceptor (PR) outer segments (OSs). Much work in the pastdecade has thus focused on the replacement of this monolayer,initially with an autologous cell source and more recently usingstem cells. Using both embryonic stem cells (ESCs) and inducedpluripotent stem cells (iPSCs), several research groups have beenable to guide differentiation into a cell that displays manycharacteristics and morphological similarities to the RPE cell. Thisapproach, coupled with extensive surgical experience from routineretinal surgery and the development of skills from experimentalretinal surgery, has made stem cell-derived RPE cells idealcandidates for transplantation into retinae for the treatment ofdegenerative diseases. These include diseases such as age-relatedmacular degeneration (AMD), Stargardt’s disease (STGD) andretinitis pigmentosa (RP), for which there are currently no curativetreatments.

There are currently several phase I clinical trials in progressusing stem cell-based therapies to treat retinal disease, and thus theeye is one of the first organs to be targeted by regenerativemedicine. The results of these trials are keenly anticipated, not onlyto see whether stem cell therapy is a viable approach but also toassess the immune tolerance of stem cell therapy. An alternative

approach to using differentiated stem cells to replace thedegenerated tissue directly is to transplant only partiallydifferentiated stem cells or even the stem cells themselves, whichcan produce trophic factors that rescue the dying cells and restorefunction. In line with this, umbilical tissue-derived stem cells,foetal stem cells and autologous bone marrow-derived stem cellsare being transplanted to the eye in an attempt to maintain a healthyretina in AMD.

In this Review, we discuss the major current issues in retinalstem cell transplantation. We first provide a brief commentary ofretinal diseases, previous surgical strategies and relevant preclinicaladvances in order to contextualise current events. We next discussthe various sources of stem cells that are being applied to treatretinal diseases. The final part of this Review outlines the futureapplications and obstacles to the ideal retinal stem cell therapy.

A brief overview of stem cellsStem cells have the ability to differentiate into several cell types butthey are also capable of indefinite self-renewal in theirundifferentiated state. Stem cells may be classified according to theirpotency: that is, the range of cell types they can differentiate into.Omnipotent stem cells can differentiate into embryonic and extra-embryonic tissue, pluripotent cells have the ability to only formembryonic tissue (ectoderm, mesoderm and endoderm), andmultipotent stem cells are able to differentiate into a limited numberof cell types, dictated by a degree of previous differentiation.

Stem cells may also be classified according to their origin. Inhumans, ESCs are harvested from the inner cell mass of theblastocyst of 5-day-old preimplantation embryos and arepluripotent (Thomson et al., 1998). Foetal stem cells are harvestedfrom foetal tissue and are considered multipotent. Adult stem cellshave also been characterised in most major organs, including theeye (Singhal et al., 2012). A rich source of adult stem cells is thebone tissue, which contains both haematopoietic stem cells (HSCs)and mesenchymal stem cells (MSCs), housed in the marrow andthe stroma, respectively. Umbilical cord tissue is another source ofmultipotent stem cells that can develop into a variety of cell types.

The therapeutic application of stem cells is based on a variety ofstrategies, the most well-known of which is cell replacementtherapy. Here, the stem cells are differentiated into the desired celltype, which is then delivered to the damaged tissue in order tointegrate and restore function. An alternative method is via aparacrine effect, whereby the transplanted stem cells secrete trophicfactors that induce the resident tissue to self-restore and proliferate(Baglio et al., 2012). Additionally, there is some evidence that stemcells may fuse with individual existing cells in order to restorefunction (Ying et al., 2002).

Retinal disease candidates for stem cell-basedregenerationThe retina is an ideal target for stem cell therapies for a number ofreasons. The eye is easily accessible and there is a wealth of

Development 140, 2576-2585 (2013) doi:10.1242/dev.092270© 2013. Published by The Company of Biologists Ltd

Stem cells in retinal regeneration: past, present and futureConor M. Ramsden1,2, Michael B. Powner1, Amanda-Jayne F. Carr1, Matthew J. K. Smart1, Lyndon da Cruz1,2

and Peter J. Coffey1,3,*

1The London Project to Cure Blindness, Division of ORBIT, Institute ofOphthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL,UK. 2NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS FoundationTrust and UCL Institute of Ophthalmology, London, EC1V 2PD, UK. 3Center for StemCell Biology and Engineering, NRI, UC Santa Barbara, CA 93106, USA.

*Author for correspondence (p.coffey@ucl.ac.uk) DEVELO

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surgical expertise in dealing with the retina, which is facilitated bythe transparent cornea. This transparency also aids clinicalassessment following intervention and allows documentation withphotography and other imaging modalities. The blood-retina barrierconfers a degree of immune privilege in the healthy eye, butwhether this is of any benefit to transplants in diseased eyesremains to be seen. Furthermore, although we are dependent onsight for a normal life, the eye is not essential to sustain life. Thisallows full characterisation for the life of the graft, even if therecipient organ continues to fail. Finally, many of the diseases ofthe retina are very well characterised.

The retina is a complex multilayered tissue that relaysinformation from light energy and converts it to electrical energythat is transmitted to the brain to build up an image of the visualenvironment. Juxtaposed against the outermost layer of the retinais the retinal pigment epithelium (RPE, Fig. 1), a support tissue forthe highly metabolic PRs of the outer retina. Light is converted toelectricity within the PRs. From here, the information is transmittedinwards via the bipolar cells and finally to the ganglion cells, whichsynapse in the superior colliculus and deliver the information in aretinotopic fashion.

The normal RPE is a polarised monolayer that is derived fromthe neuroepithelium of the anterior neural plate in the developingembryo. It is a highly metabolically active cell layer with numerousand varied functions that act to support the fragile PR cell layer.The primary functions of the RPE include the transport of nutrients,recycling of retinol, production of pigment and the phagocytosis ofcone and rod OSs. The polarised RPE secretes pigment epithelium-

derived growth factor (PEDF) from its apical side and vascularendothelial growth factor (VEGF) from its basal side. Tightjunctions between neighbouring cells form the blood-retinal barrierand each cell adheres to Bruch’s membrane (Strauss, 2005).

The PR and associated retinal circuitry is indeed a complexsystem that would be challenging to re-engineer throughregenerative medicine. A much more straightforward target that hasemerged in recent years is the RPE (Strauss, 2005). There areseveral diseases that affect the RPE, leading to RPE damage(Fig. 1B) and hence loss of vision. A detailed discussion of all ofthese RPE degenerations lies outside the scope of this Review andthe focus here will be on AMD, STGD and RP, as these are the firsttargets of stem cell therapy in the retina.

Age-related macular degenerationAge-related macular degeneration is the leading cause ofirreversible blindness in the over 55 age group worldwide (de Jong,2006). Damage to the macular area of the central retina results inloss of the central visual field (Fig. 2A,B). Advanced AMD maybe labelled as dry (geographic atrophy, GA) or wet (choroidalneovascularization, CNV) based on the absence or presence,respectively, of fluid at the macula. Almost two-thirds of over 80-year olds have at least early signs of AMD. In the UK alone, thereare half a million people with late AMD and 1.75 million in theUSA, highlighting the massive burden of disease that is set toalmost double in the next 10 years (Owen et al., 2012; Friedman etal., 2004). Furthermore, once late AMD has been diagnosed in oneeye, up to 42% of patients develop late AMD in the fellow eye, and12% will be registered blind within 5 years (Jager et al., 2008).

AMD is a complex degenerative disease with a polygenichereditary component (Haines et al., 2005; Churchill et al., 2006).It arises as the result of chronic, low grade inflammation in thecentral outer retina, which leads to degeneration of the RPE and itsbasement membrane, Bruch’s membrane (Jager et al., 2008)(Fig. 1B). Under normal conditions, each RPE cell is responsiblefor the phagocytosis of millions of PR OS discs (Sparrow et al.,2010). Over time, the incomplete digestion of each phagosomeresults in accumulation of the lysosomal protein lipofuscin, whichis toxic to RPE cells. In addition, Bruch’s membrane doubles inthickness between 10 and 90 years of age, slowing the import ofnutrients and the export of waste from the RPE, and impairing theability of the RPE to adhere to its basement membrane (de Jong,2006). In the case of dry AMD, this gradual deterioration of RPEhealth leads to subsequent PR loss at the macula. In wet AMD,fluid accumulation occurs as the result of unwanted neovascularmembranes growing from the choroid through Bruch’s membraneand the RPE into the subretinal space and occasionally through theretina. This neovascularisation is driven by the presence of excessVEGF on the apical side of the RPE, which promotes the growthof fenestrated, leaky capillaries that allow the build up of fluid.Occasionally, these fragile vessels cause haemorrhage at themacula, which may result in scarring (Jager et al., 2008).

Stargardt’s diseaseStargardt’s disease is an inherited disease of the retina. It is themost common cause of macular disease in children, with anincidence of 1:10,000 live births, and it presents in the first andsecond decades of life. The disease progresses to leave subjectslegally blind as adults as the central vision fades (Fig. 2A,B). Theclinical picture is of yellow flecks in the macular region thatcorrespond to the abnormal build up of the waste pigmentlipofuscin in the PRs, and subsequently the RPE, after PR

A Healthy RPE

B Degenerated RPE

OS

OS

Ph

RPE

RPE

BM

BM

Fig. 1. Healthy and degenerated retinal pigment epithelium. (A) Healthy retinal pigment epithelium (RPE) also showing the outersegments (OS, grey) of the photoreceptors. (B) Degenerated RPE isdiscontinuous, shows the loss of adherence (yellow bars) to Bruch’smembrane (BM, green) and the loss of tight junctions (red bars) betweenRPE cells. Photoreceptor loss also occurs due, in part, to the inability ofthe degenerated RPE to phagocytose the photoreceptor outer segments,as illustrated by the lack of phagosomes (Ph) within the RPE cells. D

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phagocytosis (Walia and Fishman, 2009). The most common formof the disease is STGD1, which is autosomal recessive. It is causedby mutations in the ABCA4 gene (Allikmets et al., 1997), whichencodes a transporter protein that is normally expressed on the PROSs. The less common forms of STGD are: STGD3, caused bymutations in the ELOVL4 gene (Zhang et al., 2001), which codesfor a protein involved in the production of fatty acids in the PRs;and STGD4, caused by defects in the PROM1 gene, which codesfor a PR transmembrane glycoprotein (Yang et al., 2008). Althoughthere is a wealth of information and valuable research into themolecular genetics of STGD, there are currently no recognisedtreatments for the disease.

Retinitis pigmentosaRetinitis pigmentosa refers to a group of inherited retinaldegenerations that mostly affect the rod visual system. There areover 100 defined genetic mutations that may lead to RP, and it maybe inherited in a dominant, recessive or X-linked fashion. As such,it has a very variable clinical course, though most patients reportproblems with night blindness and progressive peripheral visualfield loss, leading to tunnel vision (Fig. 2C), which is oftenfollowed by blindness (Hartong et al., 2006). In many cases, RPprogresses to involve the central visual field. The classical clinicalpicture is of pigment deposition in the peripheral retina. Of the 1in 4000 people affected, the most common RP subtype occurs dueto mutations in the gene encoding rhodopsin and accounts for 30%of autosomal dominant cases. Although many of RP mutationscode for genes in PRs, many RP subtypes begin with primaryfailure of the RPE. The RPE-specific genes RPE65 (Hamel et al.,1994) and MERTK (Gal et al., 2000), among many others, are alsoimplicated in RP and could be ideal targets for replacement withstem cell-derived RPE.

Retinal transplantation in AMD: proof of principleCurrently, there is no treatment that can reverse dry AMD,although dietary supplementation with defined vitamins andantioxidants has been shown to slow progression (Age-Related EyeDisease Study Research Group, 2001). In the case of wet AMD,intravitreal injection of anti-VEGF agents can stabilise the disease,halting the decline of visual acuity in 90% of individuals with asubtype of wet AMD (Martin et al., 2011; Rosenfeld et al., 2006).However, this treatment is expensive and must be administeredfrequently in order to have any positive effect. Furthermore, thelong-term side-effects and potential benefits of this relatively newtherapy are yet to be elucidated.

With no curative treatments available for either type of AMD,several surgical approaches to reverse the pathology have beenattempted over the past 20 years. Surgical techniques were firstdeveloped while performing submacular surgery to removeneovascular membranes and haemorrhage. A systematic review(Giansanti et al., 2009) suggested that submacular surgery was of

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no benefit in wet AMD and, as with all procedures requiring avitrectomy, there was an increased rate of cataract, retinaldetachment and proliferative vitreoretinopathy.

Given that the diseased RPE is a major component of AMD,attempts have been made to replace the RPE at the macula, eitherby moving the macula to the non-diseased periphery or by graftingnew RPE under the macula. Sources of RPE cells that have beenused in these procedures include rotated pedicled flaps ofperipheral RPE-choroid, autologous free RPE-choroid grafts, sheetsof foetal RPE and cell suspensions of peripheral RPE (Binder et al.,2007; da Cruz et al., 2007; Chen et al., 2009; van Zeeburg et al.,2012). In the last approach, one of the major drawbacks is thatthere is no guarantee that the cells in suspension can first attach tothe diseased Bruch’s membrane (Tsukahara et al., 2002). Moreover,those cells that attach often do not form the desired monolayer thatis required for optimal RPE function, and they instead clump intorosettes (Vugler et al., 2008) or undergo anoikis (Tezel et al., 2004),a form of apoptosis specific to anchorage-dependent cells that aredissociated from their usual extracellular matrix.

Macular translocation and RPE transplantation are complex,lengthy and expensive procedures that often require further surgeryto address complications such as unplanned retinal detachment,cataracts and double vision. It is unlikely that these procedures willprove cost effective in combatting the huge burden of AMD. Afurther constraint of autologous RPE transplant is that it is difficultto collect enough material to repopulate the entire maculaadequately. Moreover, the cells being harvested are the same ageas the cells they are intended to replace. It is estimated that themacula harbours 60,000 RPE cells that potentially need replacing.To overcome this, it will be necessary to develop new techniquesto derive and expand RPE cells in vitro, or to use the paracrineeffects of stem cells to trigger rejuvenation.

Potential sources of cells to treat retinal diseaseThe ideal cell-based therapy for RPE regeneration would have highefficacy, a low failure rate, be reproducible across a range ofphenotypes and not require immunosuppression. It is preferablethat the tissue is easy to propagate, requiring a low harvest rate andwith few ethical issues. It should also cost little and require fewresources, and both the culture and transplantation of the tissueshould not be technically difficult to execute. Potential sources ofsuch cells for cellular therapy of AMD are wide ranging andinclude both pluripotent stem cells and multipotent stem cells offoetal and adult tissues.

Human embryonic stem cellsThe first account of the rescue of visual function by primate ESCs(by replacement of the RPE monolayer) was that of cynomolgusmonkey ESC-derived RPE transplanted into Royal College ofSurgeon rats (the RCS rat, see Box 1), an accepted animal modelfor AMD (Haruta et al., 2004). Since this time, two main

A Healthy visual field B Macular dysfunction C Peripheral field loss Fig. 2. The effects of retinal pigmentepithelium damage on visual field. (A) Thevisual fields in normal subjects. (B) The visualfield in the case of macular diseases, such as age-related macular degeneration and Stargardt’sdisease. (C) The tunnel visual field experienced inindividuals with retinitis pigmentosa.

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approaches to propagate RPE-like cells from human ESCs (hESCs)have been described, either spontaneous or directed. The formermethod allows the overgrowth of stem cell colonies, which resultsin spontaneous differentiation of RPE cells following removal offibroblast growth factor (FGF) from the culture medium(Klimanskaya et al., 2004; Lund et al., 2006; Vugler et al., 2008).After a few weeks, pigmented areas of cells develop, from whichthe RPE-like cells are derived. Spontaneous differentiation to RPE-like cells can also be achieved using embryoid bodies grown insuspension that are plated out as adherent colonies in neuraldifferentiation medium (Meyer et al., 2009).

A more-directed approach can be achieved by blocking the Wntand Nodal signalling pathways with dickkopf 1 (DKK1) and left-right determination factor (LEFTY) (Ikeda et al., 2005),respectively. Alternative methods include incubation sequentiallywith nicotinamide and then activin A (Idelson et al., 2009).Although yield and speed are greater with the directed methods ofRPE differentiation, there may be benefits of using a morespontaneous method. Exogenous factors used to drive stem cellsdown a neural and eye field pathway are expensive, and theassociated cell culture techniques are laborious. Furthermore,factors such as DKK1 and LEFTY are often derived from bacterialand animal sources, which can add a layer of complexity to theregulatory process for human therapies.

Human ESC-derived RPE appears to replicate very closely theendogenous tissue morphology and function. Analysis of thehESC-derived RPE grown as a monolayer revealed a carpet ofhexagonal cells with apical microvilli and pigment-containingmelanosome granules (Fig. 3). Furthermore, the hESC-derivedRPE cells displayed polarity with the correct apical orientation ofNa+K+ATPase and expressed proteins associated with tightjunctions (Vugler et al., 2008). Gene and protein expressionanalysis of the hESC-derived RPE in vitro showed expression ofproteins involved in retinol cycling, as well as expression ofRPE65, a protein specific to RPE cells. In addition, hESC-derivedRPE cells express PAX6 at levels similar to foetal RPE cells

(Klimanskaya et al., 2004); furthermore, PEDF is preferentiallysecreted from their apical surface and the tight junction protein 1(ZO1) is expressed only on their cell borders (Zhu et al., 2011).Finally, phagocytosis was observed in assays in vitro and followingtransplantation into RCS rat eyes (Carr et al., 2009; Idelson et al.,2009; Vugler et al., 2008), confirming that these cells are indeedRPE like.

With the aim of translating these studies into clinical trials,hESC-derived RPE has been transplanted into the subretinal spaceof RCS rats and has been shown to slow the degeneration of PRs,improve visual performance and sustain objective measures ofretinal electrical activity (Lu et al., 2009; Lund et al., 2006; Vugleret al., 2008). To assess safety, hESC-derived RPE has also beentransplanted into NIH III mice, which are immune deficient, anddevoid of natural killer cells and mature T and B cells (Lu et al.,2009). These mice will normally display a reduced ability to fightoff infection and combat cancer, and are thus ideal candidates forassessing tumour formation. Of the 45 animals that received atransplant, none developed teratomas or displayed any otherevidence of dysplasia throughout their life. This is encouraging,although caution must be exercised as different groups havereported tumour formation in up to 50% of mice that receivedhESC-derived neural precursors transplanted to the subretinal space(Arnhold et al., 2004). In this case, however, the neural precursorcells are only partially differentiated and thus may be moretumourigenic than the terminally differentiated hESC-derived RPEcells. Although RPE can be cultured and expanded in vitro (Saleroet al., 2012), in a healthy human macula it is generally regarded tobe post mitotic. As pluripotent stem cell-derived RPE cells displaymany structural and functional features of native RPE, it isexpected that they will not proliferate, especially if transplanted intheir natural monolayer morphology.

In summary, hESC-derived RPE cells display many of thecharacteristics of native RPE, including morphology, polarity,phagocytosis, tight junctions and retinol cycling. Aftertransplantation into animal models of AMD, hESC-derived RPEcells can rescue visual parameters and could represent the idealsource for cell therapy for AMD.

The preliminary results of a clinical trial in which a suspensionof dissociated hESC-derived RPE cells was transplanted to two

Box 1. The RCS ratThe Royal College of Surgeons (RCS) rat is a commonly used animalmodel for retinal pigment epithelium (RPE) diseases, including age-related macular degeneration (AMD), and is phenotypicallycharacterised by the build up of phagosomes on Bruch’smembrane, akin to AMD. These rats harbour a mutation in theirMertk gene (D’Cruz et al., 2000), which encodes MERTK, atransmembrane tyrosine kinase that is expressed in the RPE andplays a role in the signal transduction pathway involved in thephagocytosis of rod outer segments (OSs). In the healthy retina, thestacked phospholipid discs of the rod OSs are renewed at the baseby 10% each day. As the discs travels towards the RPE, theygenerate toxic reactive oxygen species owing to their environmentof rapid metabolism and high UV light. These increasingly unstableOS discs are phagocytosed in a circadian rhythm by the RPE. Thisevent is facilitated by adhesion through the apically expressed αVβ5integrin protein and subsequent transduction to initiate thephagocytosis process via MERTK.

Extrapolating data from RCS rats into humans is difficult. Ratshave a much lower density of cones and entirely lack maculae.Additionally, the RCS rat has a young Bruch’s membrane, unlikeAMD pathology. Indeed, experiments in which retinal detachmentsin RCS rats have been induced by saline alone have rescued somevisual response in these rats, presumably by ‘cleaning’ the debrisfrom the RPE/Bruch’s complex (Sauvé et al., 2002).

ZO1/connexin 43

Nuc

BA

Fig. 3. Phenotypic characterisation of human embryonic stem cell-derived retinal pigment epithelium. (A) Electron microscopy of humanembryonic stem cell (hESC)-derived retinal pigment epithelium (RPE)showing features typical of native polarised RPE, such as basal nucleus(Nuc) location, presence of melanocytes (white arrowhead) and apicalmicrovilli (black arrow). (B) Immunocytochemistry showing thepigmented monolayer of hESC-derived RPE cells with hexagonal shape,as demarcated by the tight junction protein ZO1 in green and theintercellular gap junction protein connexion 43 in red. D

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individuals (one with dry AMD and one with STGD) were reportedin 2012 by Advanced Cell Technology (ACT), California, USA(Schwartz et al., 2012). The cells, developed by ACT and termedMA09-hRPE, were injected into the submacular space following avitrectomy procedure. Four months after transplantation, there wasno evidence of teratoma formation and no loss of vision in eitherindividual. It was suggested that new pigmentation near theinjection site in the patient with STGD was evidence of the RPEfunction. More results are awaited from this multicentre trial (seeTable 1 for a summary of this and other trials using stem cells forretinal disease).

ESC-derived RPE cells have been shown to have themorphology and essential function of native RPE, and may be anideal source of cells for transplantation. Their source is, however,ethically contentious; thus, other types of stem cells are beingconsidered for retinal repair in AMD and other retinaldegenerations. Although replacement of the degenerated RPE is aworthwhile strategy when this layer has been lost, in earlier formsof the disease the simple rescue of the failing, but still present, RPEmay be enough to reverse pathology.

Human foetal stem cellsFoetal stem cells are derived from a wide variety of embryonic andextra-embryonic tissues and are regarded as distinct from adultstem cells and ESCs (Pappa and Anagnou, 2009). Recently, humanfoetal neural stem cells were isolated from enzymaticallydissociated brain tissue of donated aborted foetuses (16-20 weeks

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gestation). The stem cells were identified by their expression of thecell-surface maker CD133 and were expanded in cell cultureconditions that had been optimised for neural cell culture to inducerosette formation. Once dissociated, these suspensions of neuralisedfoetal stem cells were injected into the subretinal space of the RCSrat (McGill et al., 2012), where they survived and migratedthroughout the retina and away from the injection site. Basic visualparameters and retinal histology were improved in the treated rats,though the exact mechanism of repair is unknown as thetransplanted cells do not adopt a retinal morphology or expressretinal markers. These cells, labelled HuCNS-SC, have beendeveloped by Stem Cells Inc. (Newark, CA, USA). More recently,Stem Cells, in partnership with the Retina Foundation of theSouthwest (Dallas, TX, USA), have initiated a clinical trial,initially in 16 patients with dry AMD, using a sub-retinal injectionof stem cells. The results of this trial are awaited.

Human umbilical tissue-derived stem cellsHuman umbilical tissue harbours multipotent stem cells that areclassified as adult stem cells. Such stem cells can be retrieved fromdonor umbilical cords through enzymatic digestion and can beexpanded in cell culture. Suspensions of these human umbilicaltissue-derived stem cells (hUTSCs) were injected subretinally into80 RCS rats (Lund et al., 2007), which resulted in better outcomesin terms of the visual parameters compared with controls. Themechanism of repair observed is thought to be paracrine, as thehUTSCs do not change their morphology and were found to secrete

Table 1. Clinical trials using stem cells for retinal disease that are currently registered on WHO clinical trial register

Disease Institution Cell source Delivery Immuno-

suppression BCVA WHO

identifier Reference

STGD and AMD (GA)

Advanced Cell Technology, USA

hESC-derived RPE

Subretinal injection of suspension post-vitrectomy

Yes: tacrolimus

20/400 NCT01469832 (Schwartz et al., 2012)

AMD University College London and Pfizer, UK

hESC-derived RPE monolayer

Subretinal on a plastic polymer patch post-vitrectomy

Intraocular steroid

N/A NCT01691261 (Carr et al., 2009)

AMD (GA) Stem Cells Inc., USA Neuralised human foetal stem cells

Subretinal Injection post-vitrectomy

Yes: unspecified

20/400 NCT01632527 (McGill et al., 2012)

AMD (GA) Janssen R&D, USA hUTSC Micro-catheter via sclera and choroid

N/A 20/200 NCT01226628 (Lund et al., 2007)

AMD (GA), RP and ischaemic retinopathy

University of Sao Paolo, Brazil

Autologous BMHSC

Intravitreal injection N/A 20/200 NCT01518127 NCT01560715 NCT01518842

(Siqueira et al., 2011)

AMD (GA), RP, RVO and DR

University of California, Davis, USA

Autologous BMHSC

Intravitreal injection N/A 20/200 NCT01736059 (Park et al., 2012)

RP Mahidol University, Thailand

BMMSC Intravitreal injection N/A 6/120 NCT01531348 N/A

DR Tehran University of Medical Sciences, Iran

Autologous BMMSC

Intravitreal injection N/A N/A IRCT201111291414N29

(Larijani et al., 2012)*

DR General Hospital of the Chinese People’s Armed Police Force, China

BMMSC N/A N/A N/A ChiCTR-TNRC-11001491

(Li et al., 2012)*

AMD and RP All India Institute of Medical Sciences, India

Autologous BMHSC

Intravitreal injection N/A 10/200 CTRI/2010/091/000639

(Kumar et al., 2012)

AMD, age-related macular degeneration; BCVA, best corrected visual acuity; BMHSC, bone marrow-derived haematopoeitic stem cell; BMMSC, bone marrow-derived mesenchymal stem cell; DR, diabetic retinopathy; GA, geographic atrophy; hESC, human embryonic stem cell; hUTSC, human umbilical tissue-derived stem cells; N/A, information not available; RP, retinitis pigmentosa; RPE, retinal pigment epithelium; RVO, retinal vein occlusion; STGD, Stargardt’s disease. *References are not specific to retinal disease. DEVELO

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high levels of brain-derived neurotrophic factor, which is known toimprove neuronal survival. Building on these findings, JanssenResearch and Development (Philadelphia, PA, USA), a researcharm of Johnson and Johnson, have started a clinical trial of ahUTSC cell line, termed CNTO 2476, in individuals with dryAMD. These cells are delivered using a catheter delivery systemthrough the white fibrous sclera and the vascular choroidal tissueto the subretinal space. There are no results from this phase I/II trialas yet.

Bone marrow-derived HSCsIn mice, it has been shown that endogenous HSCs migrate to thesubretinal space in damaged retinae (Li et al., 2006), presumablyto initiate repair. Allogenic bone marrow-derived HSCs (BMHSCs)have been delivered via the tail vein to mice shortly afterexperimentally induced damage to the retina (Atmaca-Sonmez etal., 2006). These exogenous cells were shown to migrate to theretina, proliferate and express RPE65 (an RPE-specific protein).This expression of RPE protein suggests that these transplantedcells are reacting appropriately to their new niche; however, theexpression of RPE65 alone is not indicative of their attaining RPEfunction. RPE cells have typical morphology and many essentialfunctions, all of which would need to be characterised in thetransplanted cells in order to be confident that this is a viable RPEreplacement. One group (Otani et al., 2004) showed improvedvisual parameters in a mouse model of RP following intravitrealinjection of such BMHSCs. In humans, intravitreal injection ofBMHSCs into three individuals with RP was shown to have noadverse side effects, although visual parameters in these cases didnot improve significantly (Siqueira et al., 2011). The same groupis currently recruiting to use this therapy on subjects with AMDand vascular retinopathies such as retinal vein occlusion anddiabetic retinopathy, both of which result in retinal ischaemia. InCalifornia, another group is recruiting for a trial that usesintravitreal injection of autologous BMHSCs to treat retinal veinocclusion, following their use in animal studies in which they weredeemed to be safe (Park et al., 2012).

Bone marrow-derived MSCsBone marrow-derived MSCs (BMMSCs) also hold promise as asource for cellular therapy in diseases of the RPE. These cells wereinjected subretinally in mouse models of RP (Arnhold et al., 2007),whereupon RPE and PR morphology appeared to improve.BMMSCs were also injected into the subretinal space of the RCSrat (Lu et al., 2010), where they were found to reverse the declineof basic visual parameters. Of note, in the RCS rat, BMMSCsdisplayed no difference in graft survival regardless ofimmunosuppression, suggesting that mesenchymal cells arepotentially less immunogenic than other sources of allogenic stemcells.

Future applications and directionsRegenerative stem cell-based therapies may become the standardmeans of treating the huge burden of severe retinal degeneration, butit is still far off routine clinical practice. Although these first trialswill help answer the most pressing question of efficacy, there aremany issues that need to be resolved. Embryonic-derived stem cellsappear to be able to form cells with the structure and many of thefunctions of RPE but their origin is an ethically contentious issue, aswith foetal brain-derived stem cells. Current methods fortransplantation using cell suspensions are not likely to develop intomonolayers and there are likely to be issues with immune rejection.

Although autologous bone marrow-derived stem cells are likely tobe less immunogenic after transplantation, it remains to be seenwhether any of these modalities have the potential to restore vision.The challenge for the future, then, is to develop an ethicallyacceptable therapy that restores sight, either by transplanting retinaltissue or by inducing retinal repair, without immunological rejection.

Transplanting hESC-derived RPE as a monolayerIn an attempt to recreate the natural anatomy of the RPE, theLondon Project to Cure Blindness (LPCB; London, UK) hasdeveloped a hESC-derived RPE cell line that can be cultured as amonolayer on a thin sheet of polymer (Carr et al., 2013). Thisapproach aims to overcome the disorganised fashion in which RPEcells adhere to Bruch’s membrane when injected as a suspension(Fig. 4). The plastic polymer is also designed to act as areplacement for the aged and thickened Bruch’s membrane, andprovides an anchor for the cells as well as aiding in surgicaldelivery. A clinical trial with this RPE patch graft is currentlyrecruiting in London.

Using iPSCsESC-like cells may also be generated from differentiated somaticcells by reprogramming with multiple factors (Takahashi andYamanaka, 2006; Fusaki et al., 2009; Warren et al., 2010; Kim etal., 2009). Forced expression of four transcription factors, Oct4,Sox2, Myc and Klf4, can reprogramme these cells to ESC-like cellswith pluripotent qualities, termed iPSCs. Other groups haveachieved similar results using different combinations oftranscriptions factors, such as Oct4, Sox2, Nanog and Lin28, whichavoids the use of Myc, a known proto-oncogene (Yu et al., 2007).

RPE has also been derived from iPSCs (Buchholz et al., 2009;Carr et al., 2009; Hirami et al., 2009). As with hESC-derived RPE,there are differing protocols used to generate RPE-like cells fromiPSCs. The iPSC-derived RPE cells form monolayers that exhibitpolarity, form tight junctions, express genes vital to the visual cycleand are able to phagocytose. Transplantation of iPSC-derived RPEinto RCS rats has been shown to improve visual function after 13weeks (Buchholz et al., 2009; Carr et al., 2009; Liao et al., 2010;Meyer et al., 2009; Osakada et al., 2009). One group in Japan(Hirami et al., 2009) is planning a clinical trial using iPSC-derivedRPE for AMD. This will be among the first clinical trial usingiPSCs in humans.

Diseases that are caused primarily by single gene defects, suchas STGD, represent a greater challenge for iPSC-based therapies.This is because iPSCs generated from a diseased individual, as wellas their differentiated progeny, will still contain the disease-causinggenetic abnormality. Strategies are being developed to geneticallycorrect iPSCs prior to differentiation and transplantation. Proof ofprinciple for this approach comes from Meyer and colleagues, whocorrected the mutation in the ornithine aminotransferase gene thatcauses gyrate atrophy in patient-derived iPSCs (Meyer et al., 2011).The genetically corrected cells were then differentiated into RPEcells, which were observed to have normal levels of ornithineaminotransferase as measured by enzyme assay, compared with theuncorrected gyrate atrophy parental iPSC line (Meyer et al., 2011).In the case of AMD, genetic manipulation may not be necessary asAMD is a polygenic disorder of systemic origin and thus no singlegene corrections would provide a total cure. Moreover, the majorinsult in AMD is wear and tear through years of low gradesystemic inflammation, and the expectation is that simplyrejuvenating the damaged tissue will be enough to ‘wind back theclock’ and alleviate the symptoms. D

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iPSC-based cellular therapies represent an exciting avenue forregenerative medicine and may herald the dawn of personalisedmedicine. iPSCs also avoid many of the ethical concerns ofembryo-derived stem cells, and furthermore may be matched to theindividual. However, there are several issues that must be fullyexamined before iPSCs may be widely used in clinical practice.Although considered as functionally equivalent to ESCs, iPSCshave been shown to harbour subtle differences in gene expressionand DNA methylation (Doi et al., 2009), and thus caution must betaken in extrapolating ESC-based therapies to iPSCs. Furthermore,there have been reports of point mutations and copy numbervariation in iPSCs (Gore et al., 2011), which raises possible safetyissues. It has also been observed that chromosomal telomeres weresignificantly shortened in iPSC lines compared with native RPE(Kokkinaki et al., 2011). Telomere length is a marker for cellnascence, with shorter telomeres being associated with aging DNA.This suggests that iPSC-based therapies may not have the intendedshelf life and more than one therapeutic episode may be required.Finally, there is concern that autologous iPSCs may beimmunogenic, though this issue has been somewhat resolved by thework of Araki and colleagues, who showed that this may only betrue for pluripotent iPSCs, and not for their terminallydifferentiated progeny (Zhao et al., 2011; Araki et al., 2013; Guhaet al., 2013). An alternative approach to the use of autologousiPSCs is the development of banks of undifferentiated cells thatcould be differentiated into the desired cell type and transplantedto human leukocyte antigen-matched recipients (Zimmermann etal., 2012)

With the unknown long-term effects coupled with the potentialforced expression of known proto-oncogene genes, caution mustbe exercised before iPSC-based treatment can be extrapolated intothe clinical environment. Furthermore, the cost of truly allogeniciPSC therapy, using current technology, would be too high toproduce these treatments on the large scale needed to meet themassive demands of a disease such as macular degeneration.

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Transplantable PRs: using stem cells as a sourcePhotoreceptor death in AMD and STGD occurs as a consequence ofdysfunction of the diseased RPE. As such, rejuvenating or replacingthe RPE is a good place to start. However, when the PRs are lost, theresulting visual deficit is permanent. This is a common occurrencein advanced AMD (wet or dry), STGD and RP. For these individuals,a potential step forward in regaining vision is to replace thedegenerated PR cells. This is a viable strategy for AMD as it hasbeen shown that ganglion cells can survive in retinae even whenthere is severe underlying PR loss (Medeiros and Curcio, 2001).However, finding a suitable source of these transplantable cells is thenext step and the limiting factor. Foetal retina is an obvious sourceof retinal precursors that have the potential to reintegrate intodegenerating retina. Retinal precursors isolated from foetal tissuehave been transplanted into the subretinal space of both mice andhuman subjects with PR loss and have improved visual behaviour inthe mice (Humayun et al., 2000; MacLaren et al., 2006; Pearson etal., 2012; Radtke et al., 2004). However, it has been demonstrated(MacLaren et al., 2006) that the only viable cells that integrated intothe mature retina and differentiated into functional rod PRs werethose that were already committed to a PR fate.

When translated from mouse models to use in humans, there aretwo major drawbacks to this approach: first, the limited supply offoetal tissue; and, second, that the equivalent foetal stage forharvesting the required cells would be after 13 weeks gestation,which leads to significant ethical concerns. Both these issues canbe significantly alleviated by using pluripotent stem cells. In 2006,Lamba et al. generated PRs from hESCs-derived embryoid bodiesgrown in neural culture media supplemented with IGF1, noggin(NOG) and DKK1 (Lamba et al., 2006). Similar protocols, someof which leave out NOG for a period (Banin et al., 2006) or addLEFTY (Mellough et al., 2012; Meyer et al., 2011; Osakada et al.,2009) have also been reported and have differing success rates.Furthermore, PRs have also been generated from mouse andhuman iPSCs (Hirami et al., 2009).

A

OS

PhRPE

BM

B

RP

E p

atc

hD

eg

en

era

ted

host R

PE

PP

C

RPE

OS

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3 mm

Fig. 4. Photoreceptor rescue using human embryonic stem cell-derived retinal pigment epithelium patches. (A) The proposed mechanism ofphotoreceptor rescue using a retinal pigment epithelium (RPE) patch graft of human embryonic stem cell (hESC)-derived RPE cultured on a plasticpolymer substrate (red bracket) that is transplanted between the native, degenerated RPE (blue bracket) and photoreceptor outer segments. (B) Thepigmented RPE monolayer on the plastic polymer, ready for implantation. (C) Histological section of a patch graft (red bracket) in a normal porcine eye,lying over the healthy native pig RPE (yellow bracket). BM, Bruch’s membrane; OS, outer segments; Ph, phagosome; PP, plastic polymer; RPE, retinalpigment epithelium.

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More recently, it was shown that culture under conditions ofrelative hypoxia with supplementation of DKK1, LEFTY, activinA and other factors significantly increased the PR yield frommouse ESCs. The relative hypoxia (2% O2) is closer to tissuenormoxia in utero, thus mimicking embryogenesis, which mayexplain the higher yield (Garita-Hernández et al., 2013).

Despite the observation that PR transplantation alone can restorevisual response in a simple mouse disease model (MacLaren et al.,2006), in order to support any PR graft in advanced age-relateddisease, it is likely that the RPE would also need to be replaced inorder to mimic the healthy environment. In line with this, it hasbeen shown that the survival of PR progenitor cells was increasedwhen co-cultured with hESC-derived RPE (Zhu et al., 2011),suggesting that dual replacement is likely to be a promisingstrategy. Furthermore, recent developments have seen theformation of entire optic cups from both mouse ESCs (Eiraku etal., 2011) and hESCs (Nakano et al., 2012) when grown asaggregates of cells in minimal media conditions. This source ofstratified neural retina and RPE in a single complex may also be apotential route to develop a dual RPE/PR graft that can be used inindividuals at later stages of the disease with severe neural retinaand RPE loss. Although it may be challenging to derive a funtionalgraft from just these aggregates, they represent an excitingopportunity in disease modelling.

ConclusionsStem cell transplantation for retinal disease is currentlytransitioning from over a decade of preclinical research to phaseI/II clinical trials. The results of these trials are keenly anticipated,not just regarding efficacy but also to elucidate the levels ofimmunosuppression required, the difference between RPEreplacement and paracrine models, and to determine which deliverymethod is preferable. Further trials are expected to help determinewhether an RPE suspension is adequate or whether an RPEmonolayer is required to rescue retinal function. Thetransplantation of iPSC-derived RPE will be one of the first clinicalapplications of iPSCs and will help to define how immunogeniciPSCs are in comparison with hESCs. In addition, furtherpreclinical research is under way to develop a source of PRs fromstem cells that can be used for neural retinal replacement. Thetherapeutic application of stem cells for retinal disease has thusbegun. Whether this approach will be an effective treatment is thefirst and most important issue to address.

AcknowledgementsThe authors thank Shazeen Hasan and Sakina Gooljar for providing images ofthe hESC-RPE patch.

FundingThis work was supported by funding from The London Project to CureBlindness; the Medical Research Council (MRC) UK; the California Institute ofRegenerative Medicine (CIRM); Fight for Sight UK; The Lincy Foundation; theMacular Society; and the National Institute for Health Research (NIHR)Biomedical Research Centre based at Moorfields Eye Hospital National HealthService (NHS) Foundation Trust and University College London Institute ofOphthalmology. The views expressed are those of the author(s) and notnecessarily those of the NHS, the NIHR or the Department of Health. Depositedin PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

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