REVIEW

The potential of stem cell research for the treatmentof neuronal damage in glaucoma

Mike O. Karl

Received: 21 January 2013 /Accepted: 23 April 2013 /Published online: 25 May 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Stem cell research offers a wide variety of ap-proaches for the advancement of our understanding of basicmechanisms of neurodegeneration and tissue regenerationand for the discovery and development of new therapeuticstrategies to prevent and restore neuronal cell loss. Similarto most other regions of our central nervous system, degen-erative diseases of the retina lead to the loss of neurons,which are not replaced. Recent work in animals has provid-ed proof-of-concept evidence for the restoration of photore-ceptor cells by cell transplantation and neuronal cellreplacement by regeneration from endogenous cell sources.However, efficient therapeutic prevention of neuronal cellloss has not been achieved. Moreover, successful cell re-placement of retinal neurons in humans, including that ofganglion cells, remains a major challenge. Future successesin the discovery and translation of neuroprotective drug andgene therapies and of cell-based regenerative therapies willdepend on a better understanding of the underlying diseasepathomechanisms. Existing stem cell and cell-reprogrammingtechnologies offer the potential to generate human retina cells,to develop specific human-cell-based retina disease models,and to open up novel therapeutic strategies. Further, we mightglean substantial knowledge from species that can or cannotregenerate their neuronal retina, in the search for new

therapeutic approaches. Thus, stem cell research will pavethe way toward clinical translation. In this review, I addresssome of the major possibilities presently on offer and specu-late about the power of stem cell research to gain furtheri n s i gh t s i n t o t he pa thomechan i sms o f r e t i n a lneurodegeneration (with special emphasis on glaucoma) andto advance our therapeutic options.

Keywords Retina . Stem cells . Degeneration .

Neuroprotection . Regeneration

AbbreviationsiPSC Induced plutipotent stem cellESC Embryonic stem cellIOP Intraocular pressureRGC Retinal ganglion cellRPE Retinal pigment epithelium

Introduction

The retina in our eyes is part of the central nervous systemand has a long-standing history as an approachable part ofthe brain (Dowling 1987). With its neural circuits, it isresponsible for converting light into a pattern of electricalimpulses that inform the brain about the visual world. Theretina has a common architecture across non-mammalianand mammalian vertebrates, with six types of neurons,including two types of light-sensitive photoreceptor cellslocated in the outer nuclear layer: cones (daytime colorvision) and rods (active sensors at a low light level).Photoreceptor signals are processed through three types ofinterneurons: horizontal cells, bipolar cells, and amacrinecells. The cell bodies of these neurons, together with theretinal major supporting cell type, the Müller glia, are locat-ed in the inner nuclear layer (for retina cell types, see Fig. 1).All information is finally received by the retinal ganglionneurons, which relay the visual information from the retina

M.O.K. is an investigator supported by research grants from theGerman Center for Neurodegenerative Diseases (DZNE) and the DFG-Center for Regenerative Therapies Dresden (CRTD), TU Dresden,Germany.

M. O. Karl (*)German Center for Neurodegenerative Diseases e.V. (DZNE),Arnoldstrasse 18/18b,01307 Dresden, Germanye-mail: mike.karl@dzne.dee-mail: mike.karl@crt-dresden.de

M. O. KarlCenter for Regenerative Therapies Dresden (CRTD),Technische Universität Dresden, Fetscherstraße 105,01307 Dresden, Germany

Cell Tissue Res (2013) 353:311–325DOI 10.1007/s00441-013-1646-2

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via their axons. Ganglion cell axons gather at the optic nervehead, form the optic nerve, exit the eye, and connect tohigher brain centers.

Retinal degeneration represents a group of blinding dis-eases that increasingly impact the health and well-being ofaffected patients and occur more frequent with increasingage (Berger et al. 2010). More than 150 genes are known,and new ones are still being discovered that cause retinaldisorders upon mutation. Most known mutations cause thedegeneration of photoreceptors. In other retinal diseases,such as glaucoma and diabetic retinopathy, damage of otherretinal neurons causes visual impairment (Almasieh et al.2012). Unfortunately, no clinical therapies are currentlyavailable first to prevent retinal neuron loss and then, iftoo late, to repair or to replace damaged retinal cells effi-ciently (Clark and Yorio 2003; Lamba et al. 2009b; Zhang etal. 2012). The development of various therapies targetingprevention, progression, and repair is thus pressing andis important in the maintenance of mobility, life quality,and independence.

An incredible research effort over the last 50 years has ledto a wealth of highly valuable discoveries of potentialneuroprotective strategies acting on the neurons themselves,but so far, none has reached the clinic, for example, therapiesto prevent cell loss by protecting cells from injury, to promotethe survival of damaged cells, and to provide trophic support

in order to reduce secondary damage (Bringmann et al. 2009;Hellstrom and Harvey 2011; Johnson et al. 2009; Lebrun-Julien and Di Polo 2008; Lipton 2001; Ohlmann and Tamm2012; Osborne et al. 1999; Ritch 2000; Schmeer et al. 2012;Sena et al. 2010; Wein and Levin 2002; Wen et al. 2012;Wilson and Di Polo 2012). To date, most of the evidence fornew therapeutic approaches is based on animals in vivo or onanimal cell culture. The translation from animal models to theclinic has been hampered by various major outstanding ques-tions. A bright future for regenerative medicine of the retinaby neuronal protection, repair, and cell replacement will mostprobably depend on a better understanding of the basic mech-anisms of disease pathogenesis, neuroprotection, regenera-tion, and cell remodeling and on techniques for performingearly and well-defined diagnosis and for monitoring andpredicting disease progression.

A brief glimpse at the frontiers of retinal research showsmajor new discoveries and technological developments.Remarkably, proof-of-principle evidence for retinal tissueengineering (Eiraku and Sasai 2012; Eiraku et al. 2011;Ikeda et al. 2005; La Torre et al. 2012; Lamba et al. 2006,2010; Meyer et al. 2009, 2011; Nakano et al. 2012; Osakadaet al. 2009a, 2009b; Phillips et al. 2012; Reh et al. 2010;Sasai et al. 2012), neuronal regeneration (Karl et al. 2008;Karl and Reh 2010, 2012; Ooto et al. 2004; Osakada et al.2007; Ueki et al. 2012), and cell replacement by the trans-plantation of retinal photoreceptors (Barber et al. 2013;Bartsch et al. 2008; Boucherie et al. 2013; Eberle et al.2011, 2012; Gust and Reh 2011; La Torre et al. 2012;Lamba et al. 2006, 2009a, 2009b, 2010; MacLaren et al.2006; Pearson et al. 2012; Tucker et al. 2011; W. Wang et al.2011; West et al. 2009, 2010, 2012) and of retinal pigmentepithelium (RPE; Carr et al. 2009; Haruta et al. 2004;Idelson et al. 2009; Lu et al. 2009; Lund et al. 2006;Rowland et al. 2012; Schwartz et al. 2012; Vugler et al.2008; N.K. Wang et al. 2010; Zhu et al. 2013) has openedup new avenues for regenerative medicine. The first pro-tocols for deriving retinal progenitor cells, defined retinalneuronal cell types, RPE, and entire retina from human stemcells have been developed. Moreover, stem-cell-derivedphotoreceptors and RPE integrate into adult mouse retinaafter transplantation. And in the retina of various species,endogenous neuroprotective mechanisms have been re-vealed that might be targeted by drug stimulation in thefuture. Further, some species, such as fish and chick, arewell known for their ability to regenerate their retina byMüller-glia-derived endogenous de novo neurogenesis.Interestingly, experimental data in rodents suggest thatMüller-glia-derived neuronal regeneration can be stimulatedto some extent. All in all, these are exciting times fordiscovering basic mechanisms of retinal diseases and fordeveloping drug- and cell-based regenerative therapies.This review is an attempt to outline some of the major

�Fig. 1 a Eye and retina. The retina is a thin sheet of neuronal tissue atthe back of the eye. All seven retinal cell types form the laminatedretina with an outer nuclear layer (rods and cones), inner nuclear layer(amacrines, horizontals, Müller glia), and ganglion cell layer. Theaqueous humor is produced by the ciliary body and exits the eye viastructures in the anterior eye chamber into the bloodstream. Intraocularpressure (eye pressure) is dependent on the production of aqueoushumor and the resistance of aqueous humor outflow. (RPE retinalpigment epithelium, RGC retinal ganglion cell, AC amacrine, BPbipolar cells, HC horizontal cells, R rod photoreceptor, C cone photo-receptor, MG Müller glia) b Stem cells and retinal development. In ourretina, all neuronal cells date back to the zygote, which is the initial cellformed by the fertilization of the female egg by the male sperm. Oncethe blastocyst has formed, an inner cell mass can be isolated, the cellsof which, when grown in culture, are termed embryonic stem cells(ESCs). When development proceeds, the resulting somatic stem cellsgive rise to three germ layers (ecto-, endo-, and mesoderm). The eyesand retina derive from one patch of cells from the ectoderm, morespecifically, the anterior part of the neural tube, a region called thediencephalon. This patch of cells, which is called the eyefield, containsmultipotent progenitors that, at some point, generate the retinal pro-genitors, which give rise to all retinal cells (types and numbers) born ina defined sequence, with ganglion cells, cones, horizontals, andamacrines arising earlier than bipolars, rods, and Müller glia. Defini-tions. A single stem cell may have the ability to divide and produce allbody cells of all germ layers, including (totipotent) or excluding(pluripotent) extra-embryonic tissues, or only a restricted subset ofsomatic cells within one germ layer (multipotent). In contrast, inducedpluripotent stem cells (iPSCs) are artificially generated in the labora-tory from a non-pluripotent cell, typically an adult somatic cell, and aresimilar to natural pluripotent ESCs

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current and potential future uses of stem-cell-based retinaresearch, with a major focus on the neuronal retina. Specialemphasis will also be placed on the potential of stem cellresearch to gain further insight into glaucoma disease and todiscover and to develop novel therapeutic strategies.

Glaucoma: a major neurodegenerative diseaseof the retina

Glaucoma is an umbrella term that summarizes neurodegen-erative diseases of the retina with the pathophysiologicalhallmark of optic nerve and retinal ganglion cell (RGC)degeneration. These diseases are caused by various etiolo-gies including the involvement of many genes and environ-mental effects (Almasieh et al. 2012). Thus, glaucoma is nota single entity but a heterogeneous collection of diseaseswith diverse pathomechanisms that all cause RGC loss.Similar to other neurodegenerative diseases of the centralnervous system, such as Alzheimer disease or Parkinsonism(Gupta and Yucel 2007), glaucoma is multifactorial andcomplex, with age as a risk factor. Glaucomas are charac-terized by a mostly slow and progressive cell loss thatcauses neuronal functional deficits and thus vision loss.The sight of the patient is reduced first at the peripheryand ultimately involves central high acuity vision. Thisdevastating disease can be found in about 1–4% of allhumans older than age 45, with an estimated 60–70 millionpatients suffering worldwide (Clark and Yorio 2003;Quigley and Broman 2006; Zhang et al. 2012). Obviously,there is a pressing need to prevent glaucoma.

A major reason for the lack of effective neuroprotectiveglaucoma therapies is that the underlying cellular and mo-lecular mechanisms are mostly still unclear. Taken together,patient and animal studies have indicated that, in glaucoma,RGC loss is connected to functional and structural impair-ment of at least three intraocular tissues: the RGCs, theoptic nerve head, and the trabecular meshwork. Numerouspathomechanisms for RGC loss have been suggested basedon experimental evidence including, but not limited to,endogenous signaling processes within RGCs and betweenRGCs and other retinal cell types. In addition, glaucomatousdamage of the optic nerve (RGC axons) is attributable tovasogenic and/or other optic nerve head changes. The detailsare beyond the scope of this review, but some key facts are:

(1) Most importantly, in all cases RGCs are progressivelylost, which causes visual impairment.

(2) Glaucomas are clinically classified by three parameters:(i) primary (idiopathic) or secondary (associated withsome other ocular or systemic conditions) glaucoma;(ii) state of the anterior chamber angle: open angle(undisturbed aqueous humor outflow; Fig. 1a and text

below) or closed angle (aqueous humor outflow pathwayis blocked) and (iii) acute or chronic state (most formsare chronic). The most prevalent forms are primary openangle glaucoma (POAG; the most prevalent form inEurope and North America) and primary angle closureglaucoma (the most prevalent form in the East Asianpopulation).

(3) Intraocular pressure (IOP) is a major risk factor forglaucoma and is regulated by aqueous humor physiol-ogy (Civan 2008). The posterior chamber of the eye ismostly filled by vitreous humor, and the remainingspaces are filled by aqueous humor. Aqueous humoris continuously secreted via the ciliary body in theposterior eye chamber and is drained through the tra-becular meshwork and uveoscleral outflow pathwaylocated at the interface of the iris root and cornea inthe anterior chamber. Importantly, increased IOP seemsneither sufficient nor necessary for the development ofPOAG but is highly associated with it. To date, insightsinto IOP regulation have been made through a vastarray of experimental techniques and clinical observa-tions. IOP reducing agents or surgical procedures arecurrently the only clinically available glaucoma treat-ments. IOP reducing drugs need to be applied daily andtopically to the eye by the patient. Whereas, in somepatients, therapeutic intervention of high IOP successfullyslows down the rate of the progression of the visualimpairments for some time, it is not effective in others.In any case, therapeutical IOP reduction does not reversefunctional damage to the retinal neurons and does not stopglaucoma progression for good.

(4) In closed-angle glaucoma, parts of the iris tissue blockthe outflow of aqueous fluid out of the trabecularnetwork in the iridocorneal angle (see Fig. 1a, bluearrow) resulting in acute or chronically increased IOP.A surgical therapeutic intervention might help to un-block the outflow and thus relieve the pressure, but along-term need of medical IOP treatment remains.

(5) Thus, the current holy grail of therapeutic targets arethe prevention and neuroprotection of retinal neuronsand cell replacement therapies, since any retinal neuronlost today can so far not be replaced.

The good news is that, because of the scientific achieve-ments over the last century, many potential neuroprotectivetargets and approaches (e.g., drugs, gene therapies, cell-based therapies) have been suggested, some of which haveshown positive effects on RGC survival in animals in vivoor in animal cell culture. Nevertheless, despite enormouswork and financial investment, no candidate to date hasemerged as providing successful neuroprotective therapyin humans (Bringmann et al. 2009; Hellstrom and Harvey2011; Johnson et al. 2009; Lebrun-Julien and Di Polo 2008;

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Lipton 2001; Ohlmann and Tamm 2012; Osborne et al.1999; Ritch 2000; Schmeer et al. 2012; Sena et al. 2010;Wein and Levin 2002; Wen et al. 2012; Wilson and Di Polo2012). A large gap still remains in the translation fromanimals to humans, so that even the most promising candi-dates have not been validated in human RGCs in the labo-ratory or in patients as yet. In glaucoma, success of anintervention might strongly depend on the timing and etiol-ogy, neither which can as yet be easily diagnosed. Further,major ethical concerns have to be considered, and the ap-propriate patient group needs to be identified before a hu-man trial of neuroprotectants (be it a drug or cell) can beperformed. Another major caveat regarding translation isthat probably none of the currently available glaucomaanimal models truly reflects the diversity or specificity ofhuman glaucoma disease (Bouhenni et al. 2012; Levin2001). The major glaucoma forms (e.g., POAG) do notoccur spontaneously in animals, and although the exper-imental induction of increased IOP or the application ofneurotoxins do indeed cause RGC loss in animals, oneneeds to keep in mind that both might differ fromhuman pathology.

Therefore, a major starting point for the development ofeffective therapies remains the better understanding of thepathogenesis of glaucoma (Almasieh et al. 2012; Bahr 2000;Farkas and Grosskreutz 2001; Quigley 1999; Wax and Tezel2002). Strikingly, genetic variance seems to play a role inthe occurrence and development of POAG suggesting thatthe genetic background not only exerts an influence, butalso harbors the primary cause. Genome-wide associationstudies and other genetic studies have identified glaucomasusceptibility loci for monogenic common POAG includingcommon single nucleotide polymorphisms and rare copynumber variants (Alward et al. 1996; Burdon 2012; Daviset al. 2011; Fan et al. 2006; Fingert et al. 2011; Gemenetzi etal. 2012; Jeck et al. 2012; Wiggs et al. 2012; Yang and Zack2011). Many of the genes identified are possibly expressedin the neuronal retina, but their functional roles in glaucomaare unknown. Further, if genetic information turns out to berelevant, this might solve an additional problem. Glaucomais often diagnosed when the patients experience vision loss,by which time the disease is at an advanced state. OnceRGCs are lost, vision cannot be recovered. RGC loss occursslowly but progressively over a long period of time, so thatoptimal treatment should be aimed at preventing any loss inthe first place. To do this, diagnostic (including genetic)tools to detect the glaucoma type and disease onset at anearly stage are needed. Additionally, clinical translationwould profit from other biomarkers to monitor glaucomaand its therapeutic success over time.

Taken together, the above discussion leads to some majorquestions. What are the pathomechanisms of glaucoma dis-eases and how many different diseases are there? How

should we monitor disease progression? How should weidentify useful drugs? How should we validate targets?Which patients and what type and stage of glaucoma re-spond to a given drug therapy? Is there one therapy to savethem all? If so, how do we find, validate, and monitortherapies successfully?

Potential applications of stem cells for retina research

In recent years, stem cell research has renewed interest andopened up major new fields in glaucoma research. In thefollowing, I will discuss some of the new opportunities thatstem cell research and technologies arising from it mightoffer toward studying disease pathomechanisms, findingnew therapeutic approaches, identifying and testingneuroprotective drugs, and facilitating translation to thehuman retina.

Stem cells in retinal development

The basic knowledge and groundwork for the field of retinastem cell research is rooted in developmental biology, and athorough review of the literature is beyond the scope of thisreview. Today, much is known about the types, the timing,and ultimately the nicely laminated pattern of cells generat-ed during retinogenesis (Agathocleous and Harris 2009;Brzezinski and Reh 2011; Cepko 1999; Sernagor et al.2012). In brief, the retina develops from a patch of cells calledthe eyefield region of the neural plate (neuroectoderm), whichis defined by the expression of eyefield transcription factors(EFTFs). EFTFs are required, and some even are sufficient,for eye formation (Graw 2010; Zuber 2010). Once the eyefieldis formed, it increases in size and becomes divided into twoareas, each giving rise to one evagination and an optic vesicle,part of which becomes the neuronal retina. Retinogenesisstarts from multipotent retinal progenitors that generate alltypes of retinal neurons and the major type of glia, namelyMüller glia (Fig. 1b). Cell birthdating and lineage tracingstudies have shown that retinal cells are born in a definedorder over time. Data suggest that a large network of under-lying transcription factors regulate retinal cell fate choice.Based on this current knowledge, the first protocols for deriv-ing retinal progenitor cells, defined retinal neuronal cell types,and RPE have been developed (Ikeda et al. 2005; La Torre etal. 2012; Lamba et al. 2006, 2010; Meyer et al. 2009, 2011;Osakada et al. 2009a, 2009b; Phillips et al. 2012; Reh et al.2010; Zhu et al. 2013). Most recently, even entire eyecup-likethree-dimensional tissue from human and mouse embryonicstem cells (ESCs) has been generated in the cell culture dish(Eiraku and Sasai 2012; Eiraku et al. 2011; Nakano et al.2012; Sasai et al. 2012). Nevertheless, the exact molecularmechanisms of cell fate determination and its potential

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regulation by extrinsic factors are not fully solved. Moreover,we do not yet know the precise way that most retinal neurontypes are further specified into many cell subtypes, e.g., ap-proximately nine types of bipolars and more than 20 typeseach of amacrine and ganglion cells. Further studies are nec-essary to determine the processes that lead to two-layeredoptic cups and a fully wired and functional retina (Mumm etal. 2005). Excitingly, the possible increasing use of humancells in studies of developmental biology might lead to novelinsights and might reduce or even replace some animal exper-iments that sometimes, on their own, are not easily translatedto humans.

Toward human-cell-based disease modeling and therapies

Major advances in developmental biology have provided thebasis for the development of protocols to generate all typesof body cells and tissues starting from pluripotent stem cellsin the cell culture dish (Figs. 2, 3). Today, increasinglysophisticated and refined protocols are being published togenerate particular cell types of choice. Improvements arestill necessary and ongoing to provide sufficient cells inlarge numbers and at high purity and full function.

Outstandingly, knowledge based on cell lines and animalstudies has been integrated so that, today, mouse and humanESCs can be used to recapitulate substantial parts ofretinogenesis. Thus, retinal progenitors and neuronal prog-eny and the entire self-formation of developing optic cupsresulting in a multilayered retina can be generated in the cellculture dish (Eiraku and Sasai 2012; Eiraku et al. 2011;Ikeda et al. 2005; La Torre et al. 2012; Lamba et al. 2006,2010; Meyer et al. 2009, 2011; Nakano et al. 2012; Osakadaet al. 2009a, 2009b; Phillips et al. 2012; Reh et al. 2010;Sasai et al. 2012). In one major approach, human ESCshave been expanded in their pluripotent state and thencultured as embryoid bodies and induced to regionalizeinto forebrain and more specifically eyefield and retinalprogenitors. Next, retinal progenitors have been grown incell-attached culture and encouraged to expand and dif-ferentiate into all retinal cell types, including RGCs. In amore recent approach, a defined number of ESCs hasbeen allowed to reaggregate in floating cell culture, inwhich they spontaneously form an eyefield, develop intoan optic cup-like structure, and subsequently generate astratified three-dimensional retina. These proof-of-principle studies support the overall possibility ofstudying human retinal neurons in a cell culture dish.What is missing to make our long sought dreams cometrue, i.e. the study of retinal development, diseasespathomechanisms, and therapeutic treatments by usinghuman retinal cells and the replacement of lost neuronsby cell transplantation? Many questions remain, threemajor aims from a long list are: (1) to find appropriate

cell sources for the safe and efficient generation ofdefined retinal cell types in high numbers, (2) to developdefined protocols in order to generate retinal cells (Fig. 3),either healthy cells for cell replacement therapy (Fig. 4b)or cells with a disease-specific genotype and phenotypefor disease modeling and drug testing, and (3) to developrobust and disease-specific assays or experimental condi-tions that enable the discovery and validation of newtherapies.

Some of these issues might be solved by the recentlyavailable stem cell technology that enables the generation ofany somatic cell type from healthy human individuals orfrom patient-derived cells in a cell culture dish. Patient-derived somatic cells, such as skin fibroblasts, can bereprogrammed by genetic manipulation in the laboratoryinto induced pluripotent stem cells (iPSCs; for a definition,see the legend to Fig. 1b). iPSCs similarly to ESCs cangenerate all somatic cell types (Bellin et al. 2012;Tiscornia et al. 2011; see Fig. 2a). Although, for the neuro-nal retina, only a few studies have been published so far onhuman iPSCs (Hirami et al. 2009; Jin et al. 2011; Lamba etal. 2010; Maclaren 2013; Mellough et al. 2012; Meyer et al.2009, 2011; Osakada et al. 2009b; Phillips et al. 2012), thefield is more advanced in other brain regions. For example,human iPSCs have been generated from skin cells of psy-chiatric patients, and cell differentiation into disease-specific-like neurons has been performed (Bellin et al.2012; Jung et al. 2012; Marchetto et al. 2011). Hence, yes,even today, large quantities of some, but not yet all, types ofhuman neurons can be generated from human iPSCs, andsome have been applied to drug testing (Koch et al. 2011;Wada et al. 2012). Thus, the general concept of human-cell-based disease models as a translation step toward clinicalstudies is well supported by experimental data, but so far hasnot been applied to neurodegenerative retinal diseases. iPSCtechnology might reduce the ethical concerns related to stemcell research, but still many major technical issues remain tobe solved. These problems include the definition of propercontrol cells for disease modeling and any other experi-ments, iPSC line-to-line differences, the impact of geneticbackground on experimental results, genomic instability,tumor formation, additional mutations that are independentof the disease mutation of the patient and that are acquiredduring iPSC generation or present in the host cells (forfurther details, see Bellin et al. 2012; Soldner and Jaenisch2012; Weissman 2012). Once iPSC technology is mastered,an interesting aspect for disease modeling might be to gen-erate neurons of human patients with any genetic back-ground and without knowing which genes contribute to thedisease state, e.g. as in some glaucomas. Alternatively,another interesting and promising means of generating hu-man disease models in the future is the introduction oftargeted genome modification in human ESCs or iPSCs

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Fig. 2 Retina progeny by cellreprogramming. a Inducedpluripotent stem cells (iPSCs,IPSC) provide an excitingexperimental platform on whichto model human diseases. Sofar, iPSCs have been generatedfrom skin and other cells ofpatients with various diseases.iPSCs are similar to embryonicstem cells and can differentiateinto all somatic cell types,including eyefield, retinalprogenitors, retinal neurons,and glia. b, c Induced retinalprogenitors (iRPC) and inducedRetinal Neurons (iRN),respectively, might begenerated by cellreprogramming in the future.Direct cell reprogrammingmight provide a shortcut incomparison with the detour viaiPSCs. Further, iRPC might bespecified to provide a moredefined source of retinal cellprogeny. iRN might also, in thefuture, be the fastest way toproduce human retinal cellsdirectly from human patients.d Adult stem cells are mastersof self-reprogramming. Someadult stem cells have thecapacity to be quiescent untilneeded. Once called, they canundergo endogenousreprogramming thereby gainingadult stem cell competence toproliferate and provide new cellprogeny for cell regenerationand, once completed, returnback to quiescence. Forexample, data in the zebrafishretina suggest that Müller gliaundergo this processphysiologically, and that, oncein a while, they generate a rodprogenitor that multiplies andgives rise to new photoreceptorneurons in the intact retina.Upon retinal damage, Müllerglia undergo damage-inducedreprogramming to gainmultipotent adult stem cellcapacity (see Fig. 5)

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directly. Various genome-editing technologies are currentlyin development (Cong et al. 2013; Ding et al. 2013a, 2013b;Hockemeyer and Jaenisch 2010; Hockemeyer et al. 2011;Mali et al. 2013; Rostovskaya et al. 2012; Saha and Jaenisch2009; Song et al. 2010; Zou et al. 2009). A caveat formodeling complex diseases, such as glaucoma, is that allthe structures involved in the disease, including the opticnerve head and vasculature, will not be easily modeled inthe culture dish.

The still new research field of induced cell reprogrammingoffers potential new routes not only for deriving cells effi-ciently and in a timely fashion, but also for novel therapeuticapproaches. Since the discovery of iPSCs, various recentpublications have suggested that fibroblasts can also be di-rectly reprogrammed into neuron-like cells (with and withoutgoing through a stem cell-like state) and into multipotentneuronal stem cells, which then might give rise to differenti-ated cell progeny (Cherry and Daley 2012; Corti et al. 2012;Karow et al. 2012; Kim et al. 2011; Lujan et al. 2012; Thier etal. 2012; Vierbuchen and Wernig 2011, 2012). Further, theidentity of postmitotic neuronal cells has been changed by invivo reprogramming in the cortex of mice, which even leads

to neuronal rewiring (De la Rossa et al. 2013; Rouaux andArlotta 2013). Ultimately, one can imagine that diverse pro-tocols will enable direct cell reprogramming efficiently togenerate defined retina cell types and numbers for cell-basedtherapies and other therapy development (including gene ther-apy testing and drug screening). Currently, no protocols haveyet been published that enable the efficient generation ofretinal progeny from another specific somatic cell type by cellreprogramming. So far, some studies suggest that retinal pro-genitors and RGCs can be generated by reprogrammingmouse iPSCs via the genes Pax6 (Suzuki et al. 2012) andAtoh7 (Chen et al. 2010), respectively. Neurogenic genes suchas Ascl1, Sox2, and other transcription factors might repro-gram RPE cells into neuron-like cells (Ma et al. 2009; Seko etal. 2012; S.Z. Wang et al. 2010) and amacrine and photore-ceptor progenitors into RGCs (Mao et al. 2013). Most recent-ly, an initial report has provided evidence for the in vivoreprogramming of one type of retinal neuron into anothersuggesting that the reprogramming of postmitotic photorecep-tor neurons might even prevent retinal degeneration (Montanaet al. 2013). The field of cell reprogramming is just at itsbeginning, and to date, induced reprogramming basically

Fig. 3 Retina disease modeling and translation. a–c Human embryon-ic stem cells (ESC); these are still the most reliable source of retinalneurons today. Recent studies show that, from mouse and humanpluripotent stem cells, small three-dimensional retina-like structures(b) and enriched retinal neuron cell cultures (c) can be generated.d With the existing technologies, one could generate defined mutationin human pluripotent stem cells in order to produce retinal progenywith a disease phenotype. e Induced reprogrammed cells; variouspotential cell sources might in the future be available to provide humanretina cells in the cell culture dish. Human induced pluripotent stem

cells (hiPSC) are available, and human induced retinal progenitors(hiRPC) and induced retinal neurons (iRN; see also Fig. 2) are potentialalternatives that might be derived by direct induced cellreprogramming from human-patient-derived donor cells in culture(e.g., skin fibroblasts). Thus, these technologies might enable us todiscover and validate potential new therapeutic approaches including(1) retinal cell replacement by cell transplantation, (2) human-cell-based disease modeling, (3) neuroprotection, and (4) cell regenerationand repair

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means the conversion of a given cell type into another by theinduction of defined genetic manipulation (such as transcrip-tion factors) or by the application of pharmaceutical modula-tors and/or other environmental factors. Studies ofdevelopmental retinogenesis have provided significant candi-dates and mechanisms, so that one can easily imagine theproduction of induced retinal cells, including progenitors orneurons, by cell reprogramming (see Fig. 2b, c). Cellreprogramming technology also holds promise to drive thedevelopment of various therapeutic strategies. For example,some specific stem cells are currently being investigated aspotential sources of (neuro)trophic factors (Fig. 4a), but again,the details are beyond the scope of this review. In the future,cell reprogramming and stem cell research might support thedevelopment of engineered defined cell-based neuroprotectivetherapeutics (Fischbach et al. 2013).

Adult stem cells and regeneration

One long-standing research topic that may teach us the way togenerate and even to regenerate a required defined cell type bycell reprogramming is that of adult stem cells. Some adultstem cells are masters of self-reprogramming (Bermingham-McDonogh and Reh 2011). For example, in zebrafish, retinalMüller glia have an adult stem cell competence and generate,lifelong and physiologically, photoreceptor neurons in lownumbers (Fig. 2d). Interestingly, upon retinal damage,Müller glia undergo regulated regenerative reprogramming,thereby gaining specific competence to generate progenitor-

like progeny that might be able to regenerate all retinal celltypes in quantities required to restore vision (Fig. 5; for areview, see Karl and Reh 2010). Thus, by studying the under-lying mechanisms, we might learn how to reprogram a givencell enabling it to achieve adult stem cell and regenerativepotential.

Transgenic technologies have enabled detailed studies ofanimal models of regeneration and the elucidation of thepotential of adult stem cells, their molecular and cellularmechanisms of cell repair, and cell replacement by endoge-nous de-novo cell genesis (Tanaka and Ferretti 2009).Studies of regeneration in various species including, butnot limited to, flatworms, axolotls, fishes, rodents, andhumans ultimately offer an enormous potential for the dis-covery of new technologies and therapeutic approaches. Thedifferences in regeneration, such as cell sources, mecha-nisms, and their limitations, between and sometimes evenwithin species are a huge source of knowledge for therapydevelopment. By studying retinal endogenous potentials forprevention, compensation, and regeneration in neurodegen-erative and age-related diseases, we might glean novelknowledge leading to the development of defined strategiesfor cell neuroprotection, replacement, repair, repro-gramming, and engineering from the regenerative programs.The ultimate goal is to make the research also applicable tothe human retina in which the loss of neurons frequentlyleads to permanent functional impairment.

Here, I provide a glimpse into the field of retinal regener-ation, with a special but reduced focus on RGCs as an

a b

EXOGENOUS APPROACH

MGRGC

HC

AC

BP

CR

EXTRINSIC FACTORS

neuroprotection

NEUROPROTECTION

damage

CELL TRANSPLANT

CELL REPLACEMENT

Fig. 4 Exogenous regenerative approach. The retina is an easilyaccessed part of our central nervous system for drug- and cell-trans-plantation-based therapy. a Neuroprotection. Neuroprotective drugs ortrophic factors could be applied by intraocular injection, topical eye-drops, and various cell-based stem cell based approaches. Stem-cell-derived trophic factors have been suggested to prevent or slow downneurodegeneration. (RGC retinal ganglion cell, AC amacrine, BP

bipolar cells, HC horizontal cells, R rod photoreceptor, C cone photo-receptor, MG Müller glia). b Cell replacement. Animal studies suggestthat a significant number of photoreceptors can structurally and func-tionally integrate into the healthy rodent retina upon transplantation.Studies for the replacement of other retinal cell types exist but are notyet as advanced compared with those for photoreceptors

Cell Tissue Res (2013) 353:311–325 319

example. Until recently, retinal regeneration was thought to belimited to lower cold-blooded vertebrates, such as fish andfrogs (Karl and Reh 2010; Lamba et al. 2009b). For example,fish and amphibian can regenerate their retinas almost per-fectly and regain a high degree of normal function.Unfortunately, mammals (including humans) do not possessthis capability, and so when the RGCs are lost in degenerativedisorders such as glaucoma, these neurons are never restored.However, a few years ago, higher vertebrates, such as birds,were also found to possess some capacity for regeneration. Inthe posthatch chick retina, several types of neurotoxic injurycause retinal degeneration, and the application of mitogensstimulates Müller-glia-derived neuronal regeneration (Fischer2005; Karl and Reh 2010).

Interestingly, in zebrafish, Müller glia are true adult stemcells that slowly divide and generate, lifelong and physio-logically, progenitor progeny that produce photoreceptors.However, upon damage, Müller glia in the fish retina re-spond to the loss of neurons by up-regulating mitotic genesto stimulate the production of a larger number of progenitorsthat are competent to provide all retinal cells (includingRGCs) in need of replacement to restore vision. Moreover,zebrafish have the capacity not only to regenerate new cells,but also to repair damaged cells, such as RGC axons(Fimbel et al. 2007; Sherpa et al. 2008). RGC axon regen-eration is another major topic that this review cannot cover,

but it is a research field in its own right and certainlyrepresent a major piece in the puzzle of achieving therapeu-tic cell replacement.

Another interesting aspect will be to determine whetherthe zebrafish possesses any endogenous mechanisms(Fig. 5a) that are not sufficiently active in rodents andhumans but that provide neuroprotection and prevent sec-ondary damage, thereby maintaining the structural integrityof the non-damaged parts of the retina.

In contrast to fish and birds, pathological conditions inthe mammalian vertebrate retina lead to changes in Müllerglia, analogous to the response of astrocytes to injury inother regions of the central nervous system, often calledreactive gliosis (for reviews, see Bringmann et al. 2009;Karl and Reh 2010; Robel et al. 2011). The role of gliosisis complex by providing neuroprotection (Fig. 5a) and repairand by being harmful by forming a glial scar (particularlythe proliferative form), or possibly both. Gliosis is correlat-ed with many changes, but whether it is always an imped-iment to tissue recovery, protection to secondary lesion, orboth remains a crucial question. Müller glia have long beenknown to respond to essentially all pathological alterationsof the human retina, including glaucoma and optic nervedamage. Similarly, experimental glaucoma (elevated IOP orneurotoxicity) or axotomy of the rodent retina induce, with-in hours, cellular (hypertrophy) and molecular changes in

Fig. 5 Endogenous regenerative potential. a Neuroprotection. Uponretinal injury, Müller glia undergo endogenous damage-inducedreprogramming that alters their molecular and cellular phenotype. Therole of gliosis is complex, and one crucial question is whether it canprovide protection to primary and secondary lesions (trophically or struc-turally) and support cellular repair in humans, and if so, in what way. Datafrom various animals suggest that Müller glia provide and are involved inmany neuroprotective mechanisms. b Retinal regeneration. In the retina,

Müller glia can be reprogrammed to gain adult stem cell competence. Inzebrafish regeneration is complete: Neuronal damage induces Müller gliacell reprogramming, cell cycle re-entry, and progenitor-like cell genera-tion; the generated cells differentiate and replace lost neurons. In com-parison to zebrafish, neuronal regeneration in chick is reduced, and inmammalian vertebrates so far some evidence suggests only possible uponexperimental stimulation

320 Cell Tissue Res (2013) 353:311–325

retinal Müller glia. Thus, so far, in humans and rodents,RGC death is neither rescued nor replaced by endogenousand spontaneous regeneration.

However, research in the last few years has shown that, inrodents, retinal regeneration can be stimulated by the appli-cation of various factors in vivo and ex vivo. Data fromseveral laboratories suggest that, depending on the species,animal age, in/ex vivo model, or genetic manipulation, atleast some types of neurons regenerate in low numbers(Bhatia et al. 2011; Giannelli et al. 2011; Harada et al.2011; Karl et al. 2008; Karl and Reh 2010, 2012; Kiyamaet al. 2012; Ooto et al. 2004; Osakada et al. 2007; Singhal etal. 2012; Takeda et al. 2008; Wan et al. 2007, 2008). Forexample, in adult mice in vivo, an intraocular injection ofneurotoxin NMDA causes the loss of most ganglion andamacrine cells. Subsequent mitogen injection stimulatessome Müller glia proliferation resulting in a small, butsignificant, amount of regenerated amacrine neuron progenyin vivo. Interestingly, a similar approach nicely inducesregeneration in the zebrafish retina (Wan et al. 2012).Future studies are aimed at elucidating and overcoming thelimitations in endogenous regeneration in mice and humans.By doing so, we might learn not only the way to utilize thesurviving cells capacity to provide therapeutic benefit, butalso whether degenerative processes hinder endogenousself-repair and neuroprotective mechanisms or even inducesecondary neuronal cell loss.

Concluding remarks

This review highlights some of the significant progress thathas been made and the potential of stem cell research in thediscovery and development of future therapies for retinaldiseases. Looking ahead, human-cell-based models and re-generative animal models have a great potential as astepping stone for regenerative medicine of the retina andits clinical translation, including glaucoma. Thus, the gener-ation of neurons from cells of patients with retinal disordersmight provide novel insights into disease pathomechanisms,disease progression, early diagnosis, drug discovery, and ther-apy validation. For glaucoma, the opportunities here include,at the very least, being able to gain access to a sufficientsupply of human RGCs and, at best, being able to investigatepathologies and to find neuroprotective therapies in humanretina models in the cell culture dish. If efficient RGC regen-eration and/or transplantation can be achieved in animals, thisnot only might open up the therapeutic option for humanpatients, but also might enable the investigation of glaucomain humanized animal models. These are exciting times fordiscovering basic mechanisms of neuronal degeneration, re-generation, neuroprotection, drug discovery, and therapytranslation.

Acknowledgment I thank Mortimer M. Civan, Marius Ader, themembers of my laboratory, colleagues at the CRTD and DZNE, andthe anonymous reviewers for critically reading the manuscript, forhelpful discussions, and for support. My apologies are extended inadvance to all colleagues whose work I could not include here. I amgrateful to colleagues in the field for their great research contributionsthat have inspired parts of this review.

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