Human embryonic stem cell applications for retinal degenerations

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Experimental Eye Research xxx (2013) 1e10

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Experimental Eye Research

journal homepage: www.elsevier .com/locate/yexer

Human embryonic stem cell applications for retinal degenerations

Joseph Reynolds, Deepak A. Lamba*

Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA

a r t i c l e i n f o

Article history:Received 12 April 2013Accepted in revised form 8 July 2013Available online xxx

Keywords:human ES cellsretinal differentiationRPEtransplantationphotoreceptorseye developmentembryonic stem cells

* Corresponding author. Tel.: þ1 415 209 2076.E-mail address: [email protected] (D.A. La

0014-4835/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.exer.2013.07.010

Please cite this article in press as: Reynolds, JResearch (2013), http://dx.doi.org/10.1016/j.

a b s t r a c t

Loss of vision in severe retinal degenerations often is a result of photoreceptor cell or retinal pigmentepithelial cell death or dysfunction. Cell replacement therapy has the potential to restore useful vision forthese individuals especially after they have lost most or all of their light-sensing cells in the eye. Areliable, well-characterized source of retinal cells will be needed for replacement purposes. Humanembryonic stem cells (ES cells) can provide an unlimited source of replacement retinal cells to take overthe function of lost cells in the eye. The author’s intent for this review is to provide an historical overviewof the field of embryonic stem cells with relation to the retina. The review will provide a quick primer onkey pathways involved in the development of the neural retina and RPE followed by a discussion of thevarious protocols out in the literature for generating these cells from non-human and human embryonicstem cells and end with in vivo application of ES cell-derived photoreceptors and RPE cells.

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1. Introduction

The retina is the light-sensing inner-most layer in the eye. Themain cells involved in light-perception are the photoreceptor cellslining the back of the neural retina. Additionally, phototransductionrequires active participation from the pigment epithelial cellsoverlying the photoreceptors. Any dysfunction of these two cell-types can lead to varying degrees of visual loss. Inherited and ac-quired retinal degenerations affect millions of people in the US andabroad. There are no effective therapies for most of these patients.Discovery of human embryonic stem cells in 1998 has revolution-ized the way we think about cell-replacement therapy. Embryonicstem cells have the potential to provide an unrestricted source ofnew cells for patients with any degenerative condition. In the pastfew years, a number of groups have shown the potential to effi-ciently guide the cells to various lineages including photoreceptorsand retinal pigment epithelial cells. In this review, we will coversome basics on embryonic stem cells and eye development. Wewilldiscuss how knowledge of basic development can be used to guideefficient protocols. Embryonic stem cells can in turn allow us tobetter understand some early human development events whichare otherwise difficult to study. In the end, we will show thetranslational value of embryonic stem cells in eye diseases.

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2. Embryonic stem cells

Embryonic stem cells are undifferentiated cells derived from theinner cell mass of the blastocyst. These cells can self-renew indef-initely under appropriate culture conditions while still maintainingpluripotency i.e. the ability to differentiate into most, if not all, cellsin the body. Embryonic stem cells provide an attractive potentialtowards cell and tissue engineering to generate replacement cellsfor various degenerative conditions including age-related maculardegeneration, diabetes mellitus, cardiovascular disorders, etc.Mouse embryonic stem cells were first isolated by two independentgroups in 1981 (Evans and Kaufman, 1981; Martin, 1981). In 1998,James Thomson’s group at Wisconsin first derived human embry-onic stem cells (Thomson et al., 1998). Since then, a number of labsall around theworld have been able to generate new stem cell lines.The current list of NIH approved human embryonic stem cell linesis maintained at the NIH Stem Cell registry (http://grants.nih.gov/stem_cells/registry/current.htm). A number of these lines are alsoavailable in GMP-grade from sources including Wicell, City of Hopeand Biotime for clinical applications.

ES cells can be maintained in an undifferentiated state usingmouse or human fibroblasts or conditioned media from these cells.There has been a recent push towards use of xeno-free chemicallydefined media. The first such media with good results was theTESR2 media by Stem Cell Technologies. Recently, it has beenshown that a minimal chemically defined media using just 8 cul-ture media components can be used to maintain the undifferenti-ated state of human ES cells (Chen et al., 2011). The key inducers of

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Fig. 1. Morphogens playing a role in eye formation. (A) Early eye-field specification is aresult of an interplay between FGF, BMP, Wnt and IGF signaling. (B) Neural retina andRPE fates are induced by secreted morphogens from surrounding microenvironment.FGF from surface ectoderm induces neural retina while WNT, Activin and BMPs fromsurrounding mesenchyme promote RPE choice.

J. Reynolds, D.A. Lamba / Experimental Eye Research xxx (2013) 1e102

pluripotency in the media include very high-levels of TGF-b (orNodal) and bFGF. Defined xeno-free media have a very importantrole in GMP manufacture of cells allowing easier approval of cellproducts at the Food and Drug Administration (FDA). It remains tobe seen if this minimal media affects specific protocols or inductionefficiencies. Undifferentiated ES cells express a unique set ofmarkers. Human ES cells are characterized by expression of a groupof the cell surfacemarkers Tra-1-60 (Tumor-related antigen), Tra-1-81, SSEA-3 (stage-specific embryonic antigen) and SSEA-4 and anumber of transcription factors including Oct4, Nanog, and Sox2.These genes have been used now to induce pluripotency in fibro-blasts and other peripheral cells in vitro (Okita et al., 2007; Yu et al.,2007). Another cell with similar characteristics are inducedpluripotent stem cells (iPS cells). These are cells derived byreprogramming peripheral cells such as fibroblasts and lympho-blasts to a pluripotent state. A companion article in this issue dis-cusses their potential for retinal differentiation and use in diseasemodeling studies.

The potential of these cells in treatment of various disorders isunlimited. Due to their property of pluripotency, they can be guidedto differentiate into any cell or tissue in the body. This will allow usto treat any number of degenerative diseases. This reviewwill focuson retinal applications of human embryonic stem cells.

3. Developmental biology as a guide to retinal specification

In this part of the review, we will summarize the key steps andmorphogens involved in eye formation. For a stem cell differenti-ation protocol, mimicking developmental steps in vitro is bound togenerate the purest cultures as has already been shown in multipleprotocols (Chen et al., 2012; Laflamme et al., 2007; Shi et al., 2012;Si-Tayeb et al., 2010). Embryonic development proceeds by signalsproduced by strategically localized organizing centers along theembryo. These signals direct adjacent cell types to differentiate andchose specific lineages. In theory, coordinated use of these mor-phogens will allow us to specifically generate any tissue in the bodyfrom embryonic stem cells. The retina develops as the lateral out-pouching from the diencephalic region of the forebrain. Beloware described steps in its specification.

The earliest step in this process is the adoption of neuro-ectodermal identity. This region along the dorsal midline of theembryo forms the neural plate which then undergoes neurulationto form the neural tube. Neural induction is a result of signals fromthe organizer region in the underlying mesoderm first described in1938 by Hans Spemann and Hilde Mangold (Spemann, 1938).Numerous studies have shown that the cells of the organizer regionsecrete BMP antagonists such as noggin, chordin, and follistatin andthat these key factors are involved in neural induction (Hemmati-Brivanlou et al., 1994; Lamb et al., 1993; Smith et al., 1993). In thechick model it has been shown that BMP antagonism alone is notsufficient for vertebrate neural induction and that FGF signaling isrequired as an early step in ectodermal neural induction (Launayet al., 1996).

Anterior specification of the neural ectoderm for head inductionis regulated by BMP antagonism in combination with other factors(Fig. 1A). One of key signaling events involved in promoting ante-rior fates is Wnt inhibition (del Barco Barrantes et al., 2003; Wilsonand Houart, 2004). Dickkopf-1 (Dkk1) is a naturally secreted Wntantagonist from the underlying visceral endoderm involved inanteriorizing the neural ectoderm. Another cell-extrinsic factorfound to play a role in head induction is the family of insulin-likegrowth factors (IGF). Over-expression of insulin-like growth fac-tors in Xenopus embryos has been shown to induce anteriorizationof embryos and formation of ectopic eyes (Pera et al., 2001;Richard-Parpaillon et al., 2002). Tyrosine kinase pathways

Please cite this article in press as: Reynolds, J., Lamba, D.A., Human embryoResearch (2013), http://dx.doi.org/10.1016/j.exer.2013.07.010

(including FGF and IGF) activate MAP kinase (MAPK) which canphosphorylate the linker region of SMADs (Pera et al., 2003). Thisinhibits nuclear transport of the SMADs and so inhibits down-stream BMP effects, thus promoting neural induction.

In vertebrates, the eyes appear as a bilateral evagination of thediencephalon. The eye-field appears first in the form of the opticpit. The continued evagination results in the optic vesicles whichcome into close contact with the overlying ectoderm. Interactionbetween the two tissues induces the formation of the lens and thecornea in the overlying ectoderm. The head of the optic vesicle thenundergoes an invagination to form a bi-layered optic cup. The outerlayer of the cup gives rise to the retinal pigment epithelium whilethe inner layer undergoes proliferation to form the multilayeredneural retina which consists of the various retinal neuronal cellsand the Müller glia. The order of the generation of the retinalneurons is relatively conserved (Sidman, 1961). Ganglion cells arethe first to differentiate. This is followed by the cone photorecep-tors and the horizontal cells, then the amacrine cells and the finalwave of differentiation consists of the rod photoreceptors, the bi-polar cells and finally the Müller glia.

The presumptive eye field is specified prior to the developmentof the optic pits in the diencephalon. This eye field specification inthe neural plate is caused by a group of transcription factorsexpressed in this region called the eye field transcription factors(EFTFs). These include Pax6, Six3, Lhx2 and Rx/Rax. Co-expressionof these genes specifies the eye in diencephalon. While we knowmuch about the EFTFs and early eye development, we know rela-tively little about the extracellular signalingmolecules that regulatethem. Various groups have looked into the role of Wnt signaling inthe initiation and regulation of the eye fields (Rasmussen et al.,2001; Cavodeassi et al., 2005). Ligands and receptors for Wntsignaling, both canonical, ß-catenin-dependent pathway and non-canonical, ß-catenin-independent pathway, are expressed at thesite of the prospective eye field. Wnt1 or Wnt8b activate the ca-nonical Wnt-b-catenin pathway and cause a reduction in the eyefields when overexpressed in Xenopus embryos by suppressing Rxand Six3 expression. On the other hand, Wnt11 activates the non-canonical pathway, and results in larger eyes in Xenopus when


J. Reynolds, D.A. Lamba / Experimental Eye Research xxx (2013) 1e10 3

overexpressed (Cavodeassi et al., 2005). Overexpression of the Wntreceptor Frizzled-3 in Xenopus results information of multipleectopic eyes via the non-canonical Wnt pathway (Rasmussen et al.,2001).

Noggin, a known inhibitor of BMP signaling, is believed to playan important role in neural induction and eye field formation(Lamb et al., 1993; Zuber et al., 2003). In animal cap assays, Noggincan induce expression of many EFTFs including Pax6, Six3, Rx, Lhx2and Optx2 (Zuber et al., 2003). In mice, Dkk1 and noggin pathwayssynergize to induce head formation during gastrulation by duallyantagonizing Wnt and BMP signaling, acting as a head organizer(del Barco Barrantes et al., 2003). Insulin-like growth factors (IGF-1)are believed to play an important role in head and eye formation(Pera et al., 2001), and achieve this by inhibition of canonical Wntsignaling (Richard-Parpaillon et al., 2002) via kermit2, which is adual IGF receptor- and Fz receptor-interacting protein (Wu et al.,2006). Shh may also play a role later in retinal development.Recent studies suggest a role of retinal ganglion cell derived Shh inretinal stem cell maintenance (Mu et al., 2005; Wang et al., 2005).They showed that specifically ablating retinal ganglion cells (RGCs)as they differentiate resulted in reduced retinal progenitor prolif-eration. Additionally, conditional knockdown of Shh in peripheralretina results in precocious differentiation. Finally, the Notch-Deltapathway are also critical for the maintenance of the retinal pro-genitor pool. Transient inhibition of the pathway using smallmolecule inhibitors drives stage-specific synchronized differentia-tion (Nelson et al., 2007).

Asmentioned earlier, the neural retina and the RPE arise from thecommon optic neuroepithelium. A number of morphogens andtranscription factors specify these fates. Two genes critical in thisdecision include MITF and CHX10 (VSX2). CHX10 induces down-regulation of MITF resulting in a neural retinal fate of the inner layerof the bilayered optic cup. FGF from the surface ectoderm has beenshown to bias a neuro-retinal fate and can even induce trans-differentiation in RPE cells by inducing CHX10 (Horsford et al., 2005;Hyer et al., 1998; Park and Hollenberg, 1989; Pittack et al., 1991).Morphogens from the surrounding mesenchyme also affect fatespecification and bias towards RPE (Fig. 1B). Activin A, a TGFb familymember, specifically induces RPE specific genes including MITF andsuppresses the pro-neural machinery (Fuhrmann et al., 2000).Additionally, BMPs, specifically Bmp4 and Bmp7 can induce RPEfates in early optic tissue and are present in the extra-ocularmesenchyme (Muller et al., 2007). Finally, the canonical Wnt-b-catenin signaling is required for differentiation of the RPE. This oc-curs by directly regulating the expression of MITF and OTX2, two keygenes involved in RPE specification (Westenskow et al., 2009).

4. Differentiation of embryonic stem cells to retinal cells

4.1. Neuro-retinal differentiation of embryonic stem cells

Initial attempts to generate retinal cells used non-specific neuraldifferentiation protocols using either bFGF, Retinoic acid (RA) or acombination of insulin, transferrin, selenium and fibronectin (ITSFn)with the hope that the cells will by default chose anterior fates or doso following co-culture or transplantation in a retinal environment(Aoki et al., 2006; Hirano et al., 2003; Meyer et al., 2004, 2006; Sugieet al., 2005; Tabata et al., 2004; Zhao et al., 2002, 2006). These at-tempts were met with limited success. Upon co-culture withneurogenic retinal tissue, some cells expressed markers of photore-ceptor precursors like Crx and Nrl but rarely expressed differentiatedphotoreceptor markers like rhodopsin and IRBP. Following in vivotransplantation into the vitreal cavity of a mouse model of neuronaland photoreceptor degeneration, the cells penetrate into the retinallayers and have neuron like morphology. The cells however did not


express any conclusive photoreceptor markers, though they didseem to promote better survival of the remaining host photorecep-tors (Meyer et al., 2004).

These methods have now been evolved by David Gamm’s groupto undergo a second round of selective sorting. Following differ-entiation in N2 supplement containing media as embryoid bodies,the cells were replated on laminin-coated plates and around 10-days later neural rosette areas with bright ring like peripheralappearance were manually picked and expanded in low attach-ment conditions (Meyer et al., 2009). They found that the neuro-spheres generated using this method expressed all early eye-fieldtranscription factors as well as differentiated into various retinalneurons including photoreceptors. They also showed that the cellsclosely modeled early retinal development. Using small moleculeFGF-inhibition resulted in a bias of cells to an RPE fate over neuralretina associated with an increase in MITF expression as predictedfrom previous early retinal/RPE fate choice studies. Additionally,Activin A also enhanced RPE choice in these floating optic vesiclelike spheres. The protocol has been validated by other groups (Zhuet al., 2013) and has been shown recently to work in xeno-freeconditions (Sridhar et al., 2013).

One of the first efficient protocols for producing neural retinafrom mouse ES cells was using the same factors normally involvedin neural and retinal induction in vivo (Ikeda et al., 2005). YoshikiSasai’s group used a serum-free embryoid body approach in com-binationwith specific inducers, lefty-A, a nodal antagonist which isknown to have a neural induction effect in animal experiments(Meno et al., 1997), dkk1, which induces anterior neural fates (delBarco Barrantes et al., 2003), and Activin A, which has a role ininducing RPE genes as well as photoreceptor differentiation (Daviset al., 2000; Fuhrmann et al., 2000). This protocol resulted in almost30% of all cells expressing Pax6, and Rx, two transcription factorsnormally found in the retinal progenitors. Though, they did findthat serum was necessary for maximal induction. These cellshowever did not undergo high-levels of spontaneous photore-ceptor differentiation. The block could however be removed uponco-culture of these cells with re-aggregated adult retinal neurons. Alarge percentage (up to 35%) of these cells expressed rhodopsin andrecoverin, markers of photoreceptors. Transplantation of these cellsonto retinal explants resulted in their integration into host retinain vitro. They then went on to show that inhibition of the Notchpathway using gamma secretase inhibitor, DAPT, can enhancephotoreceptor differentiation (Osakada et al., 2008). Additionally,certain other factors including FGFs, taurine, Shh and retinoic acidcan further enhance rod-specific differentiation. The group alsorecently showed that some of the morphogens can be replaced bysmall molecules affecting similar pathways (Osakada et al., 2009b).

One of the first directed differentiation protocols for humanembryonic stem cells to generate highly pure cultures of retinalcells came out of Tom Reh’s lab (Lamba et al., 2006). They based theprotocol on morphogens involved the key steps in neurogenesisand anterior neural induction. Using a mixture of noggin, dkk-1,insulin-like growth factor-1 (IGF-1) and basic-fibroblast growthfactor (bFGF) in serum-free media, they could efficiently inducehuman ES cells (w80% of all cells) to take up retinal progenitor fateand express various EFTFs by three weeks. These cells upon furthermaturation underwent multi-lineage differentiation to all types ofretinal neurons including rod and cone photoreceptors (Fig. 2). Thedifferentiation was enhanced upon co-culture with mouse retinasfrom photoreceptor degenerated mice. The cells also underwentfunctional maturation in vitro responding to glutamate and NMDAas well as expressing synaptic makers in the axonal projections.This protocol has been used successfully by a number of othergroups in mouse and human pluripotent stem cells as well (Baeet al., 2012; Hambright et al., 2012; Tucker et al., 2011).


Fig. 2. Neuro-retinal differentiation of human ES cells. (A) Neural rosettes differentiating within weeks for induction with Dkk1, IGF and Noggin. (B) Optic cup-like morphology ofhuman ES cell-derived retinal tissue upon free-floating culture. (CeF) Neuro-retinal and RPE differentiation of a colony showing Pax6 (green), Otx2 (red) co-expressing RPE cells,Pax6 only expressing neuro-retinal stem cells, and differentiated tuj1 (Blue) expressing retinal neurons projecting from neuro-retinal region of the colony. (GeN) Differentiatedretinal cells expressing Brn3 (G), Hu C/D (H), Otx2 (I), Prdm1 (J), AIPL1 (K), Green cone opsin (L), Recoverin (M) and GFAP (N).


The group has recently published detailed genomic profilingdata comparing the human ES cell-derived retinal cells to fetalhuman retina (Lamba and Reh, 2011). The authors performedmicroarray analysis on ES cell-derived cells from two different time


points following retinal induction (3 weeks and 9 weeks) and onhuman fetal retinas from three different ages (60, 80, and 96 daysafter conception) allowing for direct comparison and staging of thecells with relation to human retinal development. Microarray



analysis confirmed that the protocol results in highly efficient dif-ferentiation of human ES cells toward a retinal lineage. There wascomplete absence of any contaminating cells from non-retinal CNS,PNS, mesodermal, endodermal fates or any undifferentiated em-bryonic stem cells. Gene expression profile showed a very strongcorrelation in expression of retinal genes between ES cell-derivedretina and human fetal retina. Another interesting finding was avery similar developmental progression of the cells in culture tothose in fetal eyes such that the gene expression of the 9 weekcultures was similar to 60-day human fetal retinas. Most otherpublished work has not carried similar gene profilingmaking directcomparison between protocols difficult. As genomic profiling is nolonger as expensive as it once was, such detailed analysis will in thefuture provide a better guide to differentiation efficiency andcontaminating cells compared to the routine analysis of a handfulof genes and proteins by immunocytochemistry and quantitativereal-time PCR.

4.1.1. Recapitulating early eye formation events using ES cellsOptic placode undergoes a very interesting series of

patterning events as described above to form a bi-layered opticcup. A couple of very interesting recent papers from the Sasaigroup has demonstrated that these events do occur in theirmouse and human ES cells cultures in vitro (Eiraku et al., 2011;Nakano et al., 2012). Using re-aggregated mouse or human EScells in presence of medium containing basement membranematrix, Matrigel and directed morphogens under low-attachment conditions, optic epithelia were efficiently gener-ated as evidenced by RX-transgene reporter expression. The RX-expressing areas then evaginated to form structures similar tooptic placode. The distal region of these subsequently underwentinvagination, forming a bi-layered optic cup-like morphology(Fig. 2B). Like in the eye, the invaginated inner epitheliumdifferentiated to neural retina, whereas the outer layer expressedpigment epithelium markers and underwent pigmentation.During the invagination stage, the authors found a local reduc-tion in phospho-myosin light chain 2 levels in the distal tipwhich they attribute to the increased flexibility and subsequentindentation. It will be interesting to see if overexpression ofphospho-myosin light chain 2 in the distal tip will prevent theindentation and have any effect on neural retina fate specifica-tion. Another interesting finding was that the process occurred inthe absence of any surface ectoderm or lens suggesting a lesser-than previously believed role of FGF signaling from these struc-tures to the optic cup morphogenesis and neural retinal induc-tion (Hyer et al., 1998; Pittack et al., 1997).

When carrying out experiments with human cells, canonicalWnt and Shh signaling were found to be critical for RPE formationand efficient induction (Nakano et al., 2012). Also, the human EScell-derived optic cups were much larger and thicker. The invag-ination process here required integrin associated signaling. Usinganti-b1 integrin-neutralizing antibody in the media, they foundthat it interfered with the invagination process. The retinal or-ganization in the manually detached optic cups also had aremarkable correlation with retinal morphogenesis. The cellsupon differentiation showed inter-kinetic nuclear migration ashas been previously shown in the zebrafish retina (Norden et al.,2009) and then laminated to almost the right layers. There wasa small discrepancy between the mouse and human ES cells. Themouse photoreceptors always migrated to the outer nuclear layer.However, a subset of human ES cell-derived photoreceptors mal-laminated to the inner retina. Thus ES cell-derived cells can pro-vide us with a great tool to study early steps in human develop-ment which have been elusive due to technical issues as well asaccess to biological material.


4.2. Retinal pigment epithelium differentiation from embryonicstem cells

One of the first reports of RPE differentiation from embryonicstem cells used the stromal cell line (PA6) as the inducer (Kawasakiet al., 2002). Using primate ES cells, they found that approximately8% of the cells expressed Pax6 and were pigmented. The samestromal cells also induced dopaminergic neural differentiation. It isunclear what secreted factor from the PA6 lines induces this dif-ferentiation. The stromal cell line had a similar effect on mouse EScells as well (Hirano et al., 2003). Aoki et al. (2006) modified thedifferentiation protocol, showing thatWnt2b signaling can increasethe production of RPE cells from these stromal cell-induced cul-tures of mouse ESCs. The first report of human ES cell differentia-tion to RPE also involved a feeder layer (Klimanskaya et al., 2004).Robert Lanza’s group allowed human ES cells to spontaneouslydifferentiate to RPE in the presence of mouse embryonic fibroblasts.The protocol involved withdrawal of bFGF from the knock-outserum replacer containing human ES cell media. This resulted inappearance of a few pigmented areas in 3e6 weeks which weremanually picked and expanded. The expanded RPE cells expressedvarious differentiated RPE cell markers including MITF, OTX2, andPAX6. The group did a very detailed transcriptomic analysis andshowed that the human ES cell-derived RPE cells were very similarto human fetal RPE. This protocol has been used by a number ofother groups with similar success in vitro (Buchholz et al., 2009;Carr et al., 2009; Liao et al., 2010; Vugler et al., 2008).

There have been a few reports of directed differentiation pro-tocols to generate RPE. Using an embryoid body approach, Idelsonet al. (2009) showed that using a combination of nicotinamide andactivin A promoted RPE specification in human ES cells. 50% of thecolonies developed pigmented areas. The cells generated by thisapproach showed all the typical characteristics of RPE differentia-tion. The mechanism by which nicotinamide acts to promote RPEspecification is not clear. RPE differentiation has also beendemonstrated using other directed differentiation protocols usingBMP/Nodal and Wnt inhibition using recombinant proteins andsmall molecules (Hirami et al., 2009; Lamba et al., 2010; Nistoret al., 2010; Osakada et al., 2009a,b) (Fig. 3). Our personal obser-vation shows that addition of bFGF in the media can inhibit RPEdifferentiation as predicted from developmental studies while alate addition of Wnt/b-catenin agonists promote RPE fate (unpub-lished work). Similarly small molecule FGF inhibitor SU5402 hasbeen demonstrated to bias human ES cell-derived optic placode-like tissue towards RPE by inducing MITF expression and inhibit-ing Chx10 expression (Meyer et al., 2009). Similarly, Chir 99021, aGSK3b inhibitor, promotes RPE differentiation in human ES cells bypromoting canonical Wnt activity (Nakano et al., 2012). A recentreport using a combination of Dkk1, IGF1, Noggin and nicotinamideto promote optic fate and then biasing an RPE decision using smallmolecule FGF inhibitor, SU5402 along with Activin A and VIPshowed a rapid differentiation over 10 days to RPE progenitor cellsand expressing mature markers in two weeks (Buchholz et al.,2013).

Detailed functional characterization of the RPE cells derived bythese approaches have also been carried out. The commonest assayhas been to look at themost basic and important function of RPE i.e.phagocytosis. These assays involve either co-culture with photo-receptors or feeding the cells photoreceptor outer segments andassaying for the disc material inside phagosomes in the RPEfollowing wash-off (Carr et al., 2009; Idelson et al., 2009). In orderto enhance RPE maturation, the cells are often cultured on lamininor fibronectin-coated cell culture inserts (Sonoda et al., 2009). Thispromotes functional maturation resulting in differential expressionof membrane proteins like NA-K-ATPase and ZO-1, development of


Fig. 3. RPE differentiation of Human ES cells. (A) Pigmented hexagonal monolayers of RPE cells expressing Otx2 (B, red), ZO-1 (C, red) and MITF (D, green). Nuclei are counter-stained with DAPI (blue).


high trans-epithelial resistance as well as differential secretion ofcytokines like VEGF and PEDF (Kokkinaki et al., 2011; Zhu et al.,2011). Another assay commonly used to assess maturation of theepithelial barrier function is trans-epithelial resistance measure-ments. Numerous studies have shown that the cells have the abilityto reach levels similar to those of normal human RPE (Zhu et al.,2011, 2013).

5. Transplantation of ES cell-derived neural retina and RPE

5.1. Transplantation and retinal repair using ES cell-derived retinalcells

Retinal cells derived from ES cells could provide an unlimitedsource of new cells for cell-replacement strategies to take over thefunction of lost cells. This could provide a new alternate treatmentfor millions of people affected with inherited or acquire photore-ceptor degenerative disorders. To validate such an approach, thetransplanted cells need to functionally integrate into the existingcircuitry. One of the earliest evidence of validity of a photoreceptorreplacement strategy involved transplanting photoreceptorsderived from early post-natal wild-type mice in rd1-mice (Kwanet al., 1999). The transplanted cells formed a new photoreceptorlayer, expressed synaptic markers and resulted in some low-lightvisual behavioral changes in mice suggestive of functional inte-gration. One of the most detailed studies on the photoreceptorreplacement strategy was presented by Robin Ali’s group(MacLaren et al., 2006). They used GFP-labeled photoreceptorsfrom different host ages and showed maximal integration isdependent on the age of host cells. Young photoreceptors are


optimal for integrating in host retinas following sub-retinal trans-plantation. Recent work has indicated that mature photoreceptorscan also integrate just as well but efficiency of integration is limiteddue to decreased survival following cell dissociation (Gust and Reh,2011). This suggests that enhancing photoreceptor survival shouldresult in efficient integration from any differentiated photorecep-tors regardless of age. Another major barrier to photoreceptorintegration is thought to be the outer limiting membrane made bythe glial end-feet. A number of strategies to disrupt it either usinganti-sense to tight junction proteins like ZO-1, chemical disruptionusing glial toxin, alpha-aminoadipic acid, simultaneous use ofMMPs, CSPG degradation or Crb1(Rd8) mutantmice have all shownsomewhat higher integration efficiencies (Johnson et al., 2010;Pearson et al., 2010; Suzuki et al., 2006; West et al., 2008). This isa very important consideration in a degenerative environment dueto glial activation and its associated effects on the tight junctions(Campbell et al., 2007; Roesch et al., 2012). A recent comparativeanalysis of mouse retina-derived photoreceptor integration invarious models of photoreceptor degeneration provided goodinsight into these issues (Barber et al., 2013). They found thatincreasing gliosis associated with degeneration resulted in worseintegration over time and disrupting the limiting membrane insome of these mice can restore integration capacity.

One of the first reports on transplantation of ES cell-derivedneural cells used retinoic acid treated mouse ES cells (Meyeret al., 2004, 2006). The cells following intra-vitreal trans-plantation showed some ability to migrate but not much photore-ceptor differentiation. The authors observed some neuro-protectiveeffects in host retina. Similar studies were repeated using human EScell-derived neural cells in rats (Banin et al., 2006). The group



transplanted the cells both intra-vitreally and in the sub-retinalspace. The results were similar to the neurally induced mouse EScells with some migration of the transplanted cells into the hostinner retina but not a lot of photoreceptor differentiation or func-tional integration.

Retinally-specified human ES cell-derived cells were used in adetailed transplantation study (Lamba et al., 2009). The cells wereeither injected into the intra-vitreal space in newborn mice or sub-retinal space in adult mice. The newborn transplantation in wild-type mice resulted in efficient integration in all layers of the eye.The study revealed an interesting result that the transplantedretinal neurons are pre-programmed to home into the right layersof the retina. The photoreceptors moved all the way to the outernuclear layer while the inner retinal neurons never moved beyondwhere they are supposed to go. Another interesting finding wasthat all integration from the vitreal side stopped around postnatalday two. This could be related to the time when a large number ofastrocytes have migrated into the retina from the optic nerve headand formed tight-junctions (Gardner et al., 1997; Watanabe andRaff, 1988). Upon subretinal transplantation in adult wild-typemice, the human ES cell-derived photoreceptors migrate into theouter nuclear layer and extend outer segments which had similarmorphology to mouse photoreceptors (Fig. 4). Interestingly, sub-retinal transplantation restricted integration to only the photore-ceptors. None of the other neurons or retinal stem cells integratedinto the host eye. Similar results were observed with rat sub-retinaltransplantations as well (unpublished observations).

The authors also transplanted cells into 2 different models ofretinal degeneration. Upon transplantation in a light-damagemodel, they observed that the photoreceptors integrated in thesite of the ONL after most the mouse photoreceptor cells were lost.There was also some evidence of neuroprotection of the hostphotoreceptors around the site of integration of human photore-ceptors. Human ES cell-derived retinal cells were also transplantedinto a mouse model of Leber Congenital Amaurosis (LCA7). TheCrx-/- mice undergo slow photoreceptor degeneration despite acomplete loss of most phototransduction genes (Furukawa et al.,1997). The mice as a result are blind from birth. In the Crx�/�mice, similar to wild-type mice, ES cell-derived photoreceptorsintegrated into the host retina following subretinal transplantation.In the absence of host photoreceptor outer segments, the trans-planted human photoreceptors could extend only rudimentaryanterior extensions. Additionally, due to the loss of the outerlimiting membrane secondary to degeneration, few non-photoreceptor cells migrated to the inner retina as well. The

Fig. 4. Transplantation of GFP-expressing human ES cell derived photorecept


group then also carried out functional analysis of the mice usingelectroretinography (ERG). They observed that the eyes thatreceived the transplanted cells had a clear response to flashes oflight as evidenced by presence of b-waves. The control un-injectedfellow eyes or eyes transplanted with non-retinal cells neverresponded to light stimulation. B-waves in the ERG are generatedby the inner retinal neurons suggesting that the transplantedphotoreceptors are conveying the visual information to host innerretinal circuitry. Histological analysis confirmed a strong correla-tion between the size of the transplanted area and the corre-sponding B-wave response. Finally, to confirm the integration ofthese cells into the retinal circuitry, they showed the expression ofsynaptic markers synaptophysin and PSD95 in the synaptic terminiof the transplanted cells. This data confirms the value of human EScell-derived photoreceptors for cell replacement therapy inphotoreceptor degenerative diseases. A recent paper looked atlong-term integration of human ES cell-derived retinal cells inmouse retinas (Hambright et al., 2012). They observed thatimmunosuppression may not be required in mice for long-termsurvival in the subretinal space. This can be attributed to the rela-tive immunoprivileged location of the sub-retinal space (Jiang et al.,1993). In transplants where the bloodebrain barrier was damagedduring the surgery, the transplanted cells were lost to rejection. Thedata though encouraging does not directly address an in vivo dis-ease environment which may not have a well preserved bloodebrain barrier and may be leaky following transplantation surgery.

5.2. ES cell-derived RPE transplantation strategies and successes

Successful RPE-transplantation has been reported by multiplegroups (Idelson et al., 2009; Lund et al., 2006; Vugler et al., 2008). Inall of these cases, the RCS rat was used. RCS rats have a defect in theRPE merTK gene resulting in cellular dysfunction and inability tophagocytose photoreceptor outer segments (Edwards and Szamier,1977; LaVail et al., 1975). As a result, the photoreceptor cells un-dergo progressive degeneration. All the above groups showed animprovement in photoreceptor survival as well an in improvementin visual function following injection of a suspension of RPE cells. Arecent study also look at long-term survival of the human ES cell-derived RPE cells (Lu et al., 2009). The cells survived for over 200days in rats and still maintained the ability to restore some visualfunction. Based on the success of these studies, FDA recentlyapproved a clinical trial by a company, Advanced Cell Technologies.Phase 1/2 clinical trial is currently on for the use of these human EScell-derived RPE cells generated by Robert Lanza’s group for

ors (green, C) in mouse retina stained for Otx2 (red, A) and DAPI (Blue).



treatment of dry macular degeneration and Stargardt’s disease. Sofar at least 13 patients have been injected with 50,000e100000cells in the subretinal space. The company published an initialreport showing some survival and absence of any untoward effectsor immunorejection 4 months after transplantation in some pa-tients (Schwartz et al., 2012). The patients are receiving twelve-week immunosuppression regimen involving tacrolimus andmycophenolate mofetil. The results were from a very small group ofpatients and very preliminary. It remains to be seen if there is along-term functional benefit from the trial.

There is a recent push to inject polarized RPE cells on substratesinstead of using a cell suspension. There are two reasons for thisapproach. One, the RPE is a highly polarized structure havingdistinct characteristics and functions of their basal and apicalmembranes and proper orientation upon transplantation is critical.The other reason behind this is that aged and damaged basementmembrane is not a very good substrate for RPE (Del Priore andTezel, 1998; Gullapalli et al., 2005). A recent study looking atattachment of human ES cell-derived RPE on aged basementmembrane showed poor resurfacing and function (Sugino et al.,2011). To overcome these hurdles, RPE cells have recently beencultured on ultra-thin parylene substrates. Parylene membraneswith submicron thickness are semipermeable to macromoleculesand are bio-compatible to human ES cell-derived RPE (Lu et al.,2012). This group then went on to show that the hybrid devicecan be injected into the subretinal space of normal rats withoutmuch loss of cells after 1 week post-injection (Hu et al., 2012). Itremains to be seen if this approach will result in visual improve-ments in RPE-dysfunction mice and rats and if such a device can beused in patients.

6. Future directions

Human ES cells are one of the most promising source ofreplacement cells for retinal degenerations. As described above, anumber of labs around the world have been able to generate thesecells at different efficiencies. The next big step to get this to theclinic is to generate standardized protocols that are amenable toGMP manufacture and up-scaling to generate the millions or bil-lions of cells needed for transplantation in patients. Another area tofocus is dual cell-replacement therapy. Disorders affecting RPE suchas Best’s disease, dry age-related macular degeneration and certainforms of Leber Congenital Amaurosis result in secondary photore-ceptor cell loss. In these patients, visual recovery will needreplacement of both types of cells. The problem may either beaddressed in twoway. One optionwill be to restore a new RPE layerfollowed by a second surgery to replace the lost photoreceptor cells.An alternate may be to develop a bilayered structure in vitro withboth photoreceptors and RPE in the right orientation and trans-planted such an organized tissue. This approach needs to be lookedinto for complex retinal diseases. Bioengineering and materialsciences will have a great deal to contribute to this issue. Bio-scaffolds may provide additional appropriate support either forpolarization of RPE or photoreceptor maturation needed for func-tional cells as well as the mold for easy transplantation.

Another issue to address is immunorejection. Rejection oftransplanted cells and subsequent loss may turn out to be a realissues in larger cohorts of patients with varied immune-backgrounds. Additionally, following retinal degeneration, there isextensive retinal remodeling which can result in alteration ofblood-retinal barrier as well as upregulation of various inflamma-tion associated proteins. These will likely increase the possibility ofrejection of the transplanted cells. More studies need to be carriedout to better understand the immune status of a degeneratedretinal environment including carrying out long term studies in


large animal models such as pigs and dogs. On the cell side, anapproach to get around this problem is the generation of banks ofES cells based on HLA-typing. Another idea is the generation tomatched iPS cell banks or making iPS cells from patientsthemselves.

7. Conclusions

Embryonic stem cells have given new life to the field of regen-erative medicine. Even though, a number of obstacles exist to theirroutine use in the clinic, remarkable progress has been made in avery short time. Better understanding of the nature of these cells aswell as the in vivo retinal environment following retinal degener-ation will allow us to generate effective therapies for treatment ofvarious forms of retinal degeneration.

Acknowledgments

The authors would like to thank Dr. Thomas Reh and variousmembers of his lab for fruitful discussions. We would like toacknowledge the California Institute for Regenerative Medicine forthe current grant support (RB4-05785).

References

Aoki, H., Hara, A., Nakagawa, S., Motohashi, T., Hirano, M., Takahashi, Y., Kunisada, T.,2006. Embryonic stem cells that differentiate into RPE cell precursors in vitrodevelop into RPE cell monolayers in vivo. Exp. Eye Res. 82, 265e274.

Bae, D., Mondragon-Teran, P., Hernandez, D., Ruban, L., Mason, C., Bhattacharya, S.S.,Veraitch, F.S., 2012. Hypoxia enhances the generation of retinal progenitor cellsfrom human induced pluripotent and embryonic stem cells. Stem Cells Dev. 21,1344e1355.

Banin, E., Obolensky, A., Idelson, M., Hemo, I., Reinhardtz, E., Pikarsky, E., Ben-Hur, T., Reubinoff, B., 2006. Retinal incorporation and differentiation of neuralprecursors derived from human embryonic stem cells. Stem Cells 24, 246e257.

Barber, A.C., Hippert, C., Duran, Y., West, E.L., Bainbridge, J.W., Warre-Cornish, K.,Luhmann, U.F., Lakowski, J., Sowden, J.C., Ali, R.R., et al., 2013. Repair of thedegenerate retina by photoreceptor transplantation. PNAS 110, 354e359.

Buchholz, D.E., Hikita, S.T., Rowland, T.J., Friedrich, A.M., Hinman, C.R., Johnson, L.V.,Clegg, D.O., 2009. Derivation of functional retinal pigmented epithelium frominduced pluripotent stem cells. Stem Cells 27, 2427e2434.

Buchholz, D.E., Pennington, B.O., Croze, R.H., Hinman, C.R., Coffey, P.J., Clegg, D.O.,2013. Rapid and efficient directed differentiation of human pluripotent stemcells into retinal pigmented epithelium. Stem Cells Transl. Med. 2, 384e393.

Campbell, M., Humphries, M., Kenna, P., Humphries, P., Brankin, B., 2007. Alteredexpression and interaction of adherens junction proteins in the developing OLMof the Rho(�/�) mouse. Exp. Eye Res. 85, 714e720.

Carr, A.J., Vugler, A., Lawrence, J., Chen, L.L., Ahmado, A., Chen, F.K., Semo, M.,Gias, C., da Cruz, L., Moore, H.D., et al., 2009. Molecular characterization andfunctional analysis of phagocytosis by human embryonic stem cell-derived RPEcells using a novel human retinal assay. Mol. Vis. 15, 283e295.

Cavodeassi, F., Carreira-Barbosa, F., Young, R.M., Concha, M.L., Allende, M.L.,Houart, C., Tada, M., Wilson, S.W., 2005. Early stages of zebrafish eye formationrequire the coordinated activity of Wnt11, Fz5, and the Wnt/beta-cateninpathway. Neuron 47 (1), 43e56.

Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga-Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., et al., 2011. Chemically definedconditions for human iPSC derivation and culture. Nat. Methods 8, 424e429.

Chen, Y.F., Tseng, C.Y., Wang, H.W., Kuo, H.C., Yang, V.W., Lee, O.K., 2012. Rapidgeneration of mature hepatocyte-like cells from human induced pluripotentstem cells by an efficient three-step protocol. Hepatology 55, 1193e1203.

Davis, A.A., Matzuk, M.M., Reh, T.A., 2000. Activin A promotes progenitor differ-entiation into photoreceptors in rodent retina. Mol. Cell Neurosci. 15, 11e21.

del Barco Barrantes, I., Davidson, G., Grone, H.J., Westphal, H., Niehrs, C., 2003. Dkk1and noggin cooperate inmammalian head induction. Genes Dev.17, 2239e2244.

Del Priore, L.V., Tezel, T.H., 1998. Reattachment rate of human retinal pigment epithe-lium to layers of human Bruch’s membrane. Arch. Ophthalmol. 116, 335e341.

Edwards, R.B., Szamier, R.B., 1977. Defective phagocytosis of isolated rod outersegments by RCS rat retinal pigment epithelium in culture. Science 197, 1001e1003.

Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K.,Adachi, T., Sasai, Y., 2011. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51e56.

Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential cellsfrom mouse embryos. Nature 292, 154e156.


http://refhub.elsevier.com/S0014-4835(13)00200-5/sref1





































































Fuhrmann, S., Levine, E.M., Reh, T.A., 2000. Extraocular mesenchyme patterns theoptic vesicle during early eye development in the embryonic chick. Develop-ment 127, 4599e4609.

Furukawa, T., Morrow, E.M., Cepko, C.L., 1997. Crx, a novel otx-like homeobox gene,shows photoreceptor-specific expression and regulates photoreceptor differ-entiation. Cell 91, 531e541.

Gardner, T.W., Lieth, E., Khin, S.A., Barber, A.J., Bonsall, D.J., Lesher, T., Rice, K.,Brennan Jr., W.A., 1997. Astrocytes increase barrier properties and ZO-1expression in retinal vascular endothelial cells. Invest. Ophthalmol. Vis. Sci.38, 2423e2427.

Gullapalli, V.K., Sugino, I.K., Van Patten, Y., Shah, S., Zarbin, M.A., 2005. ImpairedRPE survival on aged submacular human Bruch’s membrane. Exp. Eye Res. 80,235e248.

Gust, J., Reh, T.A., 2011. Adult donor rod photoreceptors integrate into the maturemouse retina. IOVS.

Hambright, D., Park, K.Y., Brooks, M., McKay, R., Swaroop, A., Nasonkin, I.O., 2012.Long-term survival and differentiation of retinal neurons derived from humanembryonic stem cell lines in un-immunosuppressed mouse retina. Mol. Vis. 18,920e936.

Hemmati-Brivanlou, A., Kelly, O.G., Melton, D.A., 1994. Follistatin, an antagonist ofactivin, is expressed in the Spemann organizer and displays direct neuralizingactivity. Cell 77, 283e295.

Hirami, Y., Osakada, F., Takahashi, K., Okita, K., Yamanaka, S., Ikeda, H.,Yoshimura, N., Takahashi, M., 2009. Generation of retinal cells from mouse andhuman induced pluripotent stem cells. Neurosci. Lett. 458, 126e131.

Hirano, M., Yamamoto, A., Yoshimura, N., Tokunaga, T., Motohashi, T., Ishizaki, K.,Yoshida, H., Okazaki, K., Yamazaki, H., Hayashi, S., et al., 2003. Generation ofstructures formed by lens and retinal cells differentiating from embryonic stemcells. Dev. Dyn. 228, 664e671.

Horsford, D.J., Nguyen, M.T., Sellar, G.C., Kothary, R., Arnheiter, H., McInnes, R.R.,2005. Chx10 repression of Mitf is required for the maintenance of mammalianneuroretinal identity. Development 132, 177e187.

Hu, Y., Liu, L., Lu, B., Zhu, D., Ribeiro, R., Diniz, B., Thomas, P.B., Ahuja, A.K.,Hinton, D.R., Tai, Y.C., et al., 2012. A novel approach for subretinal implantationof ultrathin substrates containing stem cell-derived retinal pigment epitheliummonolayer. Ophthalmic Res. 48, 186e191.

Hyer, J., Mima, T., Mikawa, T., 1998. FGF1 patterns the optic vesicle by directing theplacement of the neural retina domain. Development 125, 869e877.

Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al., 2009. Directeddifferentiation of human embryonic stem cells into functional retinal pigmentepithelium cells. Cell Stem Cell 5, 396e408.

Ikeda, H., Osakada, F., Watanabe, K., Mizuseki, K., Haraguchi, T., Miyoshi, H.,Kamiya, D., Honda, Y., Sasai, N., Yoshimura, N., et al., 2005. Generation of Rxþ/Pax6þ neural retinal precursors from embryonic stem cells. PNAS 102, 11331e11336.

Jiang, L.Q., Jorquera, M., Streilein, J.W., 1993. Subretinal space and vitreous cavity asimmunologically privileged sites for retinal allografts. Invest. Ophthalmol. Vis.Sci. 34, 3347e3354.

Johnson, T.V., Bull, N.D., Martin, K.R., 2010. Identification of barriers toretinal engraftment of transplanted stem cells. Invest. Ophthalmol. Vis. Sci. 51,960e970.

Kawasaki, H., Suemori, H., Mizuseki, K., Watanabe, K., Urano, F., Ichinose, H.,Haruta, M., Takahashi, M., Yoshikawa, K., Nishikawa, S., et al., 2002. Generationof dopaminergic neurons and pigmented epithelia from primate ES cells bystromal cell-derived inducing activity. PNAS 99, 1580e1585.

Klimanskaya, I., Hipp, J., Rezai, K.A., West, M., Atala, A., Lanza, R., 2004. Derivationand comparative assessment of retinal pigment epithelium from human em-bryonic stem cells using transcriptomics. Cloning Stem Cells 6, 217e245.

Kokkinaki, M., Sahibzada, N., Golestaneh, N., 2011. Human induced pluripotentstem-derived retinal pigment epithelium (RPE) cells exhibit ion transport,membrane potential, polarized vascular endothelial growth factor secretion,and gene expression pattern similar to native RPE. Stem Cells 29, 825e835.

Kwan, A.S., Wang, S., Lund, R.D., 1999. Photoreceptor layer reconstruction in a ro-dent model of retinal degeneration. Exp. Neurol. 159, 21e33.

Laflamme, M.A., Chen, K.Y., Naumova, A.V., Muskheli, V., Fugate, J.A., Dupras, S.K.,Reinecke, H., Xu, C., Hassanipour, M., Police, S., et al., 2007. Cardiomyocytesderived from human embryonic stem cells in pro-survival factors enhancefunction of infarcted rat hearts. Nat. Biotechnol. 25, 1015e1024.

Lamb, T.M., Knecht, A.K., Smith, W.C., Stachel, S.E., Economides, A.N., Stahl, N.,Yancopolous, G.D., Harland, R.M., 1993. Neural induction by the secreted poly-peptide noggin. Science 262, 713e718.

Lamba, D.A., Gust, J., Reh, T.A., 2009. Transplantation of human embryonic stemcell-derived photoreceptors restores some visual function in Crx-deficient mice.Cell Stem Cell 4, 73e79.

Lamba, D.A., Karl, M.O., Ware, C.B., Reh, T.A., 2006. Efficient generation of retinalprogenitor cells from human embryonic stem cells. PNAS 103, 12769e12774.

Lamba, D.A., McUsic, A., Hirata, R.K., Wang, P.R., Russell, D., Reh, T.A., 2010. Gener-ation, purification and transplantation of photoreceptors derived from humaninduced pluripotent stem cells. PLoS One 5, e8763.

Lamba, D.A., Reh, T.A., 2011. Microarray characterization of human embryonic stemcellederived retinal cultures. IOVS 52, 4897e4906.

Launay, C., Fromentoux, V., Shi, D.L., Boucaut, J.C., 1996. A truncated FGF receptorblocks neural induction by endogenous Xenopus inducers. Development 122,869e880.


LaVail, M.M., Sidman, R.L., Gerhardt, C.O., 1975. Congenic strains of RCS rats withinherited retinal dystrophy. J. Hered. 66, 242e244.

Liao, J.L., Yu, J., Huang, K., Hu, J., Diemer, T., Ma, Z., Dvash, T., Yang, X.J.,Travis, G.H., Williams, D.S., et al., 2010. Molecular signature of primaryretinal pigment epithelium and stem-cell-derived RPE cells. Hum. Mol.Genet. 19, 4229e4238.

Lu, B., Malcuit, C., Wang, S., Girman, S., Francis, P., Lemieux, L., Lanza, R., Lund, R.,2009. Long-term safety and function of RPE from human embryonic stem cellsin preclinical models of macular degeneration. Stem Cells 27, 2126e2135.

Lu, B., Zhu, D., Hinton, D., Humayun, M.S., Tai, Y.C., 2012. Mesh-supported submi-cron parylene-C membranes for culturing retinal pigment epithelial cells. Bio-med. Microdevices 14, 659e667.

Lund, R.D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B., Girman, S.,Bischoff, N., Sauve, Y., Lanza, R., 2006. Human embryonic stem cell-derived cellsrescue visual function in dystrophic RCS rats. Cloning Stem Cells 8, 189e199.

MacLaren, R.E., Pearson, R.A., MacNeil, A., Douglas, R.H., Salt, T.E., Akimoto, M.,Swaroop, A., Sowden, J.C., Ali, R.R., 2006. Retinal repair by transplantation ofphotoreceptor precursors. Nature 444, 203e207.

Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryoscultured in medium conditioned by teratocarcinoma stem cells. PNAS 78,7634e7638.

Meno, C., Ito, Y., Saijoh, Y., Matsuda, Y., Tashiro, K., Kuhara, S., Hamada, H., 1997.Two closely-related left-right asymmetrically expressed genes, lefty-1 andlefty-2: their distinct expression domains, chromosomal linkage and directneuralizing activity in Xenopus embryos. Genes Cells Devoted Mol. Cell.Mech. 2, 513e524.

Meyer, J.S., Katz, M.L., Maruniak, J.A., Kirk, M.D., 2004. Neural differentiation ofmouse embryonic stem cells in vitro and after transplantation into eyes ofmutant mice with rapid retinal degeneration. Brain Res. 1014, 131e144.

Meyer, J.S., Katz, M.L., Maruniak, J.A., Kirk, M.D., 2006. Embryonic stem cell-derivedneural progenitors incorporate into degenerating retina and enhance survival ofhost photoreceptors. Stem Cells 24, 274e283.

Meyer, J.S., Shearer, R.L., Capowski, E.E., Wright, L.S., Wallace, K.A., McMillan, E.L.,Zhang, S.C., Gamm, D.M., 2009. Modeling early retinal development with hu-man embryonic and induced pluripotent stem cells. PNAS 106, 16698e16703.

Mu, X., Fu, X., Sun, H., Liang, S., Maeda, H., Frishman, L.J., Klein, W.H., 2005. Ganglioncells are required for normal progenitor- cell proliferation but not cell-fatedetermination or patterning in the developing mouse retina. Curr. Biol. 15,525e530.

Muller, F., Rohrer, H., Vogel-Hopker, A., 2007. Bone morphogenetic proteins specifythe retinal pigment epithelium in the chick embryo. Development 134, 3483e3493.

Nakano, T., Ando, S., Takata, N., Kawada, M., Muguruma, K., Sekiguchi, K., Saito, K.,Yonemura, S., Eiraku, M., Sasai, Y., 2012. Self-formation of optic cups and stor-able stratified neural retina from human ESCs. Cell Stem Cell 10, 771e785.

Nelson, B.R., Hartman, B.H., Georgi, S.A., Lan, M.S., Reh, T.A., 2007. Transient inac-tivation of Notch signaling synchronizes differentiation of neural progenitorcells. Dev. Biol. 304, 479e498.

Nistor, G., Seiler, M.J., Yan, F., Ferguson, D., Keirstead, H.S., 2010. Three-dimensionalearly retinal progenitor 3D tissue constructs derived from human embryonicstem cells. J. Neurosci. Methods 190, 63e70.

Norden, C., Young, S., Link, B.A., Harris, W.A., 2009. Actomyosin is the main driver ofinterkinetic nuclear migration in the retina. Cell 138, 1195e1208.

Okita, K., Ichisaka, T., Yamanaka, S., 2007. Generation of germline-competentinduced pluripotent stem cells. Nature 448, 313e317.

Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N.,Akaike, A., Sasai, Y., Takahashi, M., 2008. Toward the generation of rod and conephotoreceptors from mouse, monkey and human embryonic stem cells. Nat.Biotechnol. 26, 215e224.

Osakada, F., Ikeda, H., Sasai, Y., Takahashi, M., 2009a. Stepwise differentiation ofpluripotent stem cells into retinal cells. Nat. Protoc. 4, 811e824.

Osakada, F., Jin, Z.B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y.,Takahashi, M., 2009b. In vitro differentiation of retinal cells from humanpluripotent stem cells by small-molecule induction. J. Cell Sci. 122, 3169e3179.

Park, C.M., Hollenberg, M.J., 1989. Basic fibroblast growth factor induces retinalregeneration in vivo. Dev. Biol. 134, 201e205.

Pearson, R.A., Barber, A.C., West, E.L., MacLaren, R.E., Duran, Y., Bainbridge, J.W.,Sowden, J.C., Ali, R.R., 2010. Targeted disruption of outer limiting membranejunctional proteins (Crb1 and ZO-1) increases integration of transplantedphotoreceptor precursors into the adult wild-type and degenerating retina. CellTransplant. 19, 487e503.

Pera, E.M., Ikeda, A., Eivers, E., De Robertis, E.M., 2003. Integration of IGF, FGF, andanti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev.,3023e3028.

Pera, E.M., Wessely, O., Li, S.Y., De Robertis, E.M., 2001. Neural and head induction byinsulin-like growth factor signals. Dev. Cell 1, 655e665.

Pittack, C., Grunwald, G.B., Reh, T.A., 1997. Fibroblast growth factors are necessaryfor neural retina but not pigmented epithelium differentiation in chick em-bryos. Development 124, 805e816.

Pittack, C., Jones, M., Reh, T.A., 1991. Basic fibroblast growth factor inducesretinal pigment epithelium to generate neural retina in vitro. Development 113,577e588.

Rasmussen, J.T., Deardorff, M.A., Tan, C., Rao, M.S., Klein, P.S., Vetter, M.L., 2001.Regulation of eye development by frizzled signaling in Xenopus. Proc. Natl.Acad. Sci. U. S. A. 98 (7), 3861e3866.



































































































































































































































Richard-Parpaillon, L., Heligon, C., Chesnel, F., Boujard, D., Philpott, A., 2002. The IGFpathway regulates head formation by inhibiting Wnt signaling in Xenopus. Dev.Biol. 244, 407e417.

Roesch, K., Stadler, M.B., Cepko, C.L., 2012. Gene expression changes within Mullerglial cells in retinitis pigmentosa. Mol. Vis. 18, 1197e1214.

Schwartz, S.D., Hubschman, J.P., Heilwell, G., Franco-Cardenas, V., Pan, C.K.,Ostrick, R.M., Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R., 2012. Embryonicstem cell trials for macular degeneration: a preliminary report. Lancet 379,713e720.

Shi, Y., Kirwan, P., Livesey, F.J., 2012. Directed differentiation of human pluripotentstem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836e1846.

Si-Tayeb, K., Noto, F.K., Nagaoka, M., Li, J., Battle, M.A., Duris, C., North, P.E., Dalton, S.,Duncan, S.A., 2010. Highly efficient generation of human hepatocyte-like cellsfrom induced pluripotent stem cells. Hepatology 51, 297e305.

Sidman, R.L., 1961. Histogenesis of the mouse retina. Studies with [3H] thymidine.In: The Structure of the Eye. Academic Press, New York.

Smith, W.C., Knecht, A.K., Wu, M., Harland, R.M., 1993. Secreted noggin proteinmimics the Spemann organizer in dorsalizing Xenopus mesoderm. Nature 361,547e549.

Sonoda, S., Spee, C., Barron, E., Ryan, S.J., Kannan, R., Hinton, D.R., 2009. A protocolfor the culture and differentiation of highly polarized human retinal pigmentepithelial cells. Nat. Protoc. 4, 662e673.

Spemann, H., 1938. Embryonic Development and Induction. Yale University Press.Sridhar, A., Steward, M.M., Meyer, J.S., 2013. Nonxenogeneic growth and retinal

differentiation of human induced pluripotent stem cells. Stem Cells Transl.Med..

Sugie, Y., Yoshikawa, M., Ouji, Y., Saito, K., Moriya, K., Ishizaka, S., Matsuura, T.,Maruoka, S., Nawa, Y., Hara, Y., 2005. Photoreceptor cells from mouse ES cells byco-culture with chick embryonic retina. Biochem. Biophys. Res. Commun. 332,241e247.

Sugino, I.K., Sun, Q., Wang, J., Nunes, C.F., Cheewatrakoolpong, N., Rapista, A.,Johnson, A.C., Malcuit, C., Klimanskaya, I., Lanza, R., et al., 2011. Comparison ofFRPE and human embryonic stem cell-derived RPE behavior on aged humanBruch’s membrane. Invest. Ophthalmol. Vis. Sci. 52, 4979e4997.

Suzuki, T., Mandai, M., Akimoto, M., Yoshimura, N., Takahashi, M., 2006. Thesimultaneous treatment of MMP-2 stimulants in retinal transplantation en-hances grafted cell migration into the host retina. Stem Cells 24, 2406e2411.

Tabata, Y., Ouchi, Y., Kamiya, H., Manabe, T., Arai, K., Watanabe, S., 2004. Specifi-cation of the retinal fate of mouse embryonic stem cells by ectopic expression ofRx/rax, a homeobox gene. Mol. Cell Biol. 24, 4513e4521.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J.,Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from humanblastocysts. Science 282, 1145e1147.


Tucker, B.A., Park, I.H., Qi, S.D., Klassen, H.J., Jiang, C., Yao, J., Redenti, S., Daley, G.Q.,Young, M.J., 2011. Transplantation of adult mouse iPS cell-derived photore-ceptor precursors restores retinal structure and function in degenerative mice.PLoS One 6, e18992.

Vugler, A., Carr, A.J., Lawrence, J., Chen, L.L., Burrell, K., Wright, A., Lundh, P.,Semo, M., Ahmado, A., Gias, C., et al., 2008. Elucidating the phenomenon ofHESC-derived RPE: anatomy of cell genesis, expansion and retinal trans-plantation. Exp. Neurol. 214, 347e361.

Wang, Y., Dakubo, G.D., Thurig, S., Mazerolle, C.J., Wallace, V.A., 2005. Retinal gan-glion cell-derived sonic hedgehog locally controls proliferation and the timingof RGC development in the embryonic mouse retina. Development 132, 5103e5113.

Watanabe, T., Raff, M.C., 1988. Retinal astrocytes are immigrants from the opticnerve. Nature 332, 834e837.

West, E.L., Pearson, R.A., Tschernutter, M., Sowden, J.C., MacLaren, R.E., Ali, R.R.,2008. Pharmacological disruption of the outer limiting membrane leads toincreased retinal integration of transplanted photoreceptor precursors. Exp. EyeRes. 86, 601e611.

Westenskow, P., Piccolo, S., Fuhrmann, S., 2009. Beta-catenin controls differentia-tion of the retinal pigment epithelium in the mouse optic cup by regulating Mitfand Otx2 expression. Development 136, 2505e2510.

Wilson, S.W., Houart, C., 2004. Early steps in the development of the forebrain. Dev.Cell 6, 167e181.

Wu, J., O’Donnell, M., Gitler, A.D., Klein, P.S., 2006. Kermit 2/XGIPC, an IGF1 receptorinteracting protein, is required for IGF signaling in Xenopus eye development.Development 133, 3651e3660.

Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S.,Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al., 2007. Induced pluripotentstem cell lines derived from human somatic cells. Science 318, 1917e1920.

Zhao, X., Liu, J., Ahmad, I., 2002. Differentiation of embryonic stem cells into retinalneurons. Biochem. Biophys. Res. Commun. 297, 177e184.

Zhao, X., Liu, J., Ahmad, I., 2006. Differentiation of embryonic stem cells to retinalcells in vitro. Methods Mol. Biol. 330, 401e416.

Zhu, D., Deng, X., Spee, C., Sonoda, S., Hsieh, C.L., Barron, E., Pera, M., Hinton, D.R.,2011. Polarized secretion of PEDF from human embryonic stem cell-derived RPEpromotes retinal progenitor cell survival. Invest. Ophthalmol. Vis. Sci. 52, 1573e1585.

Zhu, Y., Carido, M., Meinhardt, A., Kurth, T., Karl, M.O., Ader, M., Tanaka, E.M., 2013.Three-dimensional neuroepithelial culture from human embryonic stem cellsand its use for quantitative conversion to retinal pigment epithelium. PLoS One8, e54552.

Zuber, M.E., Gestri, G., Viczian, A.S., Barsacchi, G., Harris, W.A., 2003. Specification ofthe vertebrate eye by a network of eye field transcription factors. Development130, 5155e5167.















































































































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Human embryonic stem cell applications for retinal degenerations