The use of stem cells in the treatment of retinal degenerative disease has received increasing attention recently.¹ This article will describe some of the key concepts behind stem cell therapy and the issues related to potential stem cell treatment in retinal degenerative diseases.

Stem cells are unspecialized cells with the capacity for unlimited self-renewal. Each daughter cell has the capacity to remain a stem cell or to differentiate into more specialized, tissue- or organ-specific cells. Human embryonic stem cells, adult stem cells and induced pluripotent stem cells are considered in detail here (See Figure 1).²

 • Human embryonic stem cells (hESCs). hESCs are derived from the inner cell mass of the blastocyst. (The inner cell mass of the three-to-five-day-old, pre-implantation-stage embryo [blastocyst] gives rise to the entire body of the organism.) hESCs are pluripotent, which means they can form all lineages of the body (i.e., ectoderm, mesoderm and endoderm). hESCs can be obtained without destruction of the embryo.³

 • Adult (somatic) stem cells. Adult stem cells typically generate the cell types of the tissue in which they reside (See Table 1). Adult stem cells are multipotent, which means they can form multiple cell types of one lineage. For example, a retinal progenitor cell can give rise to photoreceptors, bipolar cells and ganglion cells but not to corneal cells. Adult stem cells are present in many organs and tissues, e.g., brain, bone marrow, teeth, heart, gut, liver, ovarian epithelium and testis. Adult stem cells reside in a specific area of each tissue, termed a “stem cell niche.”^4,5 Adult stem cells may remain quiescent for long periods until activated by a normal need for more cells to maintain tissues, or by disease or injury. 

Figure 1. Pathways to pluripotency. Although pluripotency is a property that exists only transiently during early development, it has been harnessed in vitro in three different ways. A. Isolation of epiblasts—by removal of the zona pellucida from supernumerary blastocysts—and their subsequent culture on mitotically inactivated mouse embryonic fibroblasts is the classical approach to the derivation of lines of human embryonic stem cells, which are necessarily allogeneic to the recipients of cell types differentiated from them. B. Isolation of the equivalent cells from cloned blastocysts, generated through the process of somatic cell nuclear transfer, gives rise to nuclear transfer embryonic stem cells, which are genetically identical to the nuclear donor in all but the mitochondrial genome. This process of “therapeutic cloning” has proven successful in mice. C. In humans, therapeutic cloning has been superseded by the advent of induced pluripotency, in which the delivery of a cocktail of transcription factors to somatic cells, such as dermal fibroblasts, reprograms them to a pluripotent state, providing a source of fully autologous induced pluripotent stem cells. Whatever their mode of derivation, all pluripotent stem cells have the capacity to differentiate into derivatives of each of the three embryonic germ layers: endoderm, ectoderm and mesoderm. (Image reproduced with permission from Fairchild.73)

 • Induced pluripotent stem cells (iPSCs). Adult (somatic) cells can be reprogrammed to an embryonic state by somatic nuclear cell transfer.¹⁰ Using different approaches, two separate groups of reasearchers showed that adult (including human) cells also can be genetically reprogrammed to an embryonic stem cell-like state by being forced to express transcription factors.^11-13 iPSCs have been generated from mouse and human somatic cells by introducing Octamer 3/4 (Oct4), sex determining region Y box–containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (c-Myc),¹² or Oct4, Sox2, Nanog, and Lin2813 using retroviruses or lentiviruses. Nuclear transfer may be more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modeling or treatment.¹⁴

Human iPSCs express stem cell markers and can produce cells from all three germ layers. Although iPSCs are pluripotent stem cells, iPSCs and ESCs do differ in some important ways (See Table 2). For example, although human iPSCs and ESCs use the same transcriptional network to generate neurons in response to a given set of morphogens, iPSCs do so with significantly reduced efficiency and increased variability.¹⁵ As noted previously,¹ iPSCs have the theoretical advantage of not being rejected by the patient from whom they are derived (vs. ESCs, unless the ESCs were harvested from the patient as an embryo), but abnormal gene expression in some cells differentiated from iPSCs (both via a retroviral and episomal approach) can induce a T-cell-dependent immune response in a syngeneic recipient.¹⁶ This response is likely due to the abnormal expression of antigens not expressed during normal development or differentiation of ESCs, leading to loss of tolerance.¹⁶ Expression of these antigens is a reflection of epigenetic differences (e.g., DNA methylation) between iPSCs and ESCs.^14,17-21 Continuous passaging of iPSCs may help attenuate these differences,²² but there are risks associated with this approach, as described below. iPSCs seem to be at greater risk for tumor formation (e.g., due to p53 suppression) than ESCs. 

It is important to note that passage of ESCs in culture can lead to alterations in the cells that may render them undesirable for cell therapy. This issue is well illustrated by X chromosome inactivation (XCI). XCI refers to repression of transcription of one of the two X chromosomes in female cells.²³ (In contrast to most genes, in which the pattern of DNA methylation is identical on both alleles, genes located on X chromosomes normally have only a single allele methylated.) Female human ESCs exhibit varying degrees of XCI.²⁴ In fact, the state of XCI can vary among subcultures of a single human ESC line.^25,26 X-inactive specific transcript (XIST) is a long, noncoding RNA, the expression of which is associated with XCI.^27-30 Class I lines have both X chromosomes active (XaXa) and can upregulate XIST during differentiation. Class II lines have one inactive X chromosome (XaXi), and class III lines have one inactive X chromosome and have lost XIST expression, although they maintain XCI. Early-passage human ESCs are more likely to be XaXa.³¹

In one study involving multiple human iPSC lines, loss of XIST expression was associated with upregulation of X-linked oncogenes, downregulation of tumor suppressor genes, accelerated growth rate in vitro, and poorer differentiation in vivo.³² (In contrast, male human iPSC lines did not overexpress oncogenes and generally resembled class II cells.) Loss of XIST expression in human iPSCs can be associated with prolonged time in culture.³³ At this time, it seems reasonable to conclude that class III female human iPSC lines should be avoided for in vivo human therapy.³²

Figure 2. Applications of disease-specific induced pluripotent stem cells. The generation of induced pluripotent stem cell lines from individuals with diseases that have a recognized genetic component may provide insight into the pathogenesis of the disease itself, while offering the potential for treatment through cell replacement therapy. The differentiation of disease-specific iPSCs into cell types associated with the disease can recapitulate aspects of the pathology in vitro, providing much-needed models of human disease. These models might greatly accelerate the process of drug discovery by enabling the efficacy and toxicity of new compounds to be tested. In cases in which the genes responsible for disease are unknown, the availability of disease-specific iPSCs might provide opportunities for their elucidation and, once corrected, the resulting iPSCs could provide a source of autologous, genetically normal cell types for replacement of tissues directly affected by the ongoing disease process. (Image reproduced with permission from Fairchild.73)

The therapeutic potential of iPSCs has been demonstrated in animal models of sickle cell anemia³⁴ and Parkinson’s disease.³⁵ However, these cells contain multiple viral vector integrations that probably make them unsuitable for human clinical trials. Genome-integrating viruses can cause insertional mutagenesis and unpredictable genetic dysfunction.^36,37 The oncogenic properties of some transcription factors (e.g., c-Myc) also create safety concerns. Furthermore, one study³⁸ has shown that iPSC-derived RPE can exhibit rapid telomere shortening, chromosomal DNA damage, increased P21 expression and growth arrest, all of which may have been the result of random viral integration in the genome. This finding is consistent with a previous report that demonstrated iPSC-derived RPE began senescing during their first passage.³⁹ Rapid senescence could limit cell survival in vivo, particularly in conditions such as age-related macular degeneration, where Bruch’s membrane has many age- and disease-related alterations that can act as death signals.⁴⁰

To improve the safety of iPSCs, modified protocols that do not require c-Myc, Sox-2, and/or Klf4 have been described.^41-45 In addition, mouse iPSCs can be created without viral vectors^46-49 via repeated transfection of two expression plasmids (i.e., non-integrating vector), one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouse embryonic fibroblasts, creating iPSCs without evidence of plasmid integration. Other vector-free methods have been used to reprogram cells to pluripotency, e.g., using modified synthetic mRNA,⁵⁰ recombinant proteins that can penetrate the plasma membrane of somatic cells,^51,52 or exposing somatic cells to ESC-conditioned media,⁵³ which may be safer than using viral vectors to induce reprogramming.⁵⁰

Human iPSCs might be used to study disease pathogenesis, for high-throughput screening to identify small molecule therapy, as well as for cell-based therapy for regenerative medicine (See Figure 2).^54,55 However, with increasing time in culture, epigenetic and transcriptional aberrations have been described in PSCs,^33,56 so one must verify that the cultured PSCs exhibit the same phenotype as the somatic tissue from the patient. A number of ocular tissues have been derived from stem cells (See Table 3).

Table 1. Examples of Adult Stem Cells  Adult Stem Cell Type Specialized Cell Type Derived from Adult Stem Cell Corneal limbal stem cells Give rise to corneal epithelium.6,7 Neural stem cells Give rise to neurons, astrocytes and oligodendrocytes. Adult Müller cells May be source of photoreceptors.8,9 Hematopoietic stem cells Give rise to red blood cells, B & T cells, natural killer cells, neutrophils, basophils, eosinophils, monocytes and macrophages. Mesenchymal stem cells Mesenchymal stem cells give rise to osteocytes, chondrocytes, adipocytes and other connective tissue cells. Digestive tract epithelial stem cells Occur in deep crypts and give rise to absorptive cells, goblet cells, paneth cells and enteroendocrine cells. Skin stem cells Occur in the basal layer of the epidermis (give rise to keratinocytes) and at the base of hair follicles (give rise to the hair follicle and to the epidermis).

The immunology of stem cell transplants has been considered elsewhere¹ and has been discussed thoroughly.⁷³ Differentiated progeny of ESCs express MHC class I antigens.^74,75 Stem cells generated by somatic cell nuclear transfer are syngeneic to the nuclear donor except for the mitochondrial genes, which are of oocyte origin⁷⁶ and are a source of minor histocompatibility antigens.⁷⁷ Disparities at the minor histocompatibility loci alone can provoke rejection of ESC-derived tissue.⁷⁸ Although iPSCs might be devoid of alloreactivity, if the iPSC harbors a genetic abnormality and if this abnormality is corrected before transplantation into the iPSC donor, then an immune response may occur.⁷³

To circumvent immune rejection of transplanted stem cells, developing banks of donor iPSCs has been proposed,^73,79 especially from individuals who are homozygous at some of the major histocompatibility (MHC) loci.⁸⁰ Because disparities at minor histocompatibility loci can provoke immune rejection, it is not clear that this approach will be useful for many patients. Although MHC matching could be supplemented with immune suppressive therapy, this approach might be accompanied by an increased risk of ESC-derived tumor formation. Other strategies that might be effective include:¹ 

 • Treatment with CD4- and CD8-specific antibodies. In preclinical models, tolerance to transplanted ESC-derived tissue can be induced using a short course of non-depleting CD4- and CD8-specific monoclonal antibodies.^78,81 Lack of donor dendritic cells in the graft may be critical for the development of tolerance with this approach. 

 • Co-transplant iPSCs with dendritic cells. iPSCs can provide both immature dendritic cells that express the alloantigens for which tolerance is required as well as the therapeutic tissue of interest.^73,82 Administration of immature dendritic cells before transplantation of therapeutic tissue might help induce tolerance to alloantigens.^83,84 Exposure of the dendritic cells to pharmacological agents or blockade of amino acid catabolism can enhance their tolerogenicity.^79,85-87 

 • Co-transplant iPSCs with mesenchymal stem cells. Mesenchymal stem cells can induce tolerance to stem cell-derived tissue grafts.^88-90 

In summary, control of the immune response is likely to be an important aspect of stem cell therapy even if iPSCs are used. As noted elsewhere,^1,91 the role of the immune suppressive nature of the subretinal space, as well as the inherent immunological properties of the transplanted tissue (e.g., photoreceptors or RPE cells) in mitigating this requirement is not clear at this time.

Table 2. Comparison of Stem Cell Types* Cell Type Advantages Disadvantages Embryonic Stem Cell   Pluripotent (can form all lineages of the body) Likely to be rejected if donor is unmatched Grown relatively easily Harbors disease-causing genes of donor Adult Stem Cell Multipotent (can form multiple cell types of one lineage) Currently, relatively hard to harvest Not rejected if transplanted into donor Harbors disease-causing genes of donor Induced Pluripotent Stem Cell Pluripotent May retain epigenetic features of cell type of origin Grown relatively easily Harbors disease-causing genes of donor Probably not rejected if transplanted into donor *Adapted from Zarbin.1

Embryonic, adult and reprogrammed (including nuclear transfer, cell fusion or genetic manipulation to create a pluripotent cell) stem cells each have advantages and disadvantages as therapeutic modalities (See Table 2). Although each of these donor cell lines, unless manipulated, harbors disease-causing genes of the donor, this fact may not have practical significance. In the case of diseases such as AMD, for example, the time needed to redevelop retinal pigment epithelium and photoreceptor damage after cell transplantation might exceed the expected life span of the recipient, who might be in the eighth or ninth decade of life.

Replacement therapy is an approach to regenerative medicine in which healthy cells replace cells that have died or are dysfunctional. For example, in retinitis pigmentosa, photoreceptors die. Replacement therapy for RP could involve transplantation of cells that can integrate with the host retina and function as photoreceptors. Replacement retinal therapy is sight-restoring. 

To be useful for cell replacement therapy, stem cells must proliferate extensively to generate sufficient quantities of material if they are intended to serve as a “universal donor.” If the stem cells are derived from the patient (e.g., iPSCs), then the requirement for extensive proliferation may be reduced considerably since these cells will serve only one recipient. Stem cells must stably differentiate into the desired cell type(s). hESC-derived RPE, for example, can spontaneously dedifferentiate to non-RPE-like cells and spontaneously redifferentiate into RPE-like cells, indicating phenotypic instability.⁶² The cultures may not retain a stable phenotype after five to eight passages. ESCs and iPSCs vary in their tendency to differentiate into cells of a given lineage.^14,39

What defines a “differentiated” RPE cell? Table 4 summarizes a number of potentially important features of differentiated RPE cells.⁹²

What defines a photoreceptor cell? Gene expression profiling has been used to determine how closely ESC-derived retinal cells resemble normal retina, the developmental stage of the ESC-derived cells (relative to fetal retinal cells), and whether there are significant contaminating non-retinal cells.⁹⁵ These studies indicate that some minimal contamination with non-retinal cells (e.g., RPE, ciliary epithelium) can occur, but that undifferentiated, pluripotent cells decline with time in culture, which may mean that a longer duration differentiation protocol may minimize the risk of teratoma formation. Some features of photoreceptor differentiation rely on interactions with surrounding cells. Interaction of photoreceptors with RPE is critical for foveal development.⁹⁶ Interaction with Müller cells via crumbs homolog 1 protein (a constituent of the zonula adherens) is important for normal outer retinal organization.⁹⁷

Table 3. Ocular Tissues Derived from Stem Cells* Stem Cell Source Derived Ocular Tissue Limbal stem cell Corneal epithelium7,57  Trabecular meshwork progenitor cell Trabecular meshwork cells58  Embryonic and/or induced pluripotent stem cells Retinal ganglion cells59-61   Retinal pigment epithelial cells62-69 Photoreceptors65-67, 70-72  *Adapted from Zarbin.1

The retinal and subretinal microenvironment can influence the differentiation and functionality of transplanted cells.^63,70,98,99 Abnormalities in Bruch’s membrane may prevent transplanted hESC-derived RPE from surviving and differentiating long-term in AMD eyes.¹⁰⁰ Bruch’s membrane is derived from mesoderm, which may mean that neither hESC- nor iPSC-derived RPE will manufacture all components of Bruch’s membrane. The recipient’s microenvironment can also influence transplanted cell survival. For example, although xenografts of human iPSC-derived RPE survive about four months in Royal College of Surgeons rats,^68,69 abnormalities in RPE of AMD eyes might prevent ESC-derived photoreceptor transplants from surviving. Typical RP is characterized by early rod photoreceptor death, and cone survival depends on rod survival.^101-103 Therefore, it might be best to transplant a mixture of rods and cones to achieve improvement in cone-mediated visual function.

Transplanted cells must integrate into the surrounding tissue for replacement therapy to succeed. Targeted disruption of glial reactivity and disruption of the outer limiting membrane may improve integration of transplanted cells.^104-106 The developmental age of the donor cells may be critical for successful integration with host retina,¹⁰⁷ but it is not clear that this is the case.¹⁰⁸ The synaptic reorganization that accompanies photoreceptor degeneration in RP109 might limit the extent of functional transplanted photoreceptor integration with the host. Presumably, if the transplanted cells have differentiated and integrated appropriately, they also will function physiologically in the host tissue. Relatively few functioning cones are needed to sustain visual acuity of 20/30.¹¹⁰

Rescue refers to the preservation and, in some cases, restoration of function of tissue that is destined to die or malfunction due to an underlying disease. Cells that mediate rescue may elaborate needed trophic factors and must not proliferate in an uncontrolled manner. Rescue therapy may be sight-restoring to the degree that dying cells, which cannot support vision, can return to normal physiological function. Degenerating photoreceptors, for example, may first lose their outer segments, and thus become inefficient transducers of light energy. Under these circumstances, if rescue therapy restores elaboration of outer segments, vision might improve.

Table 4. Some Features of Differentiated RPE Cells92*  Category Feature DNA 154 "signature" genes have been defined from studies of native fetal RPE, cultured fetal RPE and native adult RPE.93  RNA MicroRNA expression is important in the induction and maintenance of stable epithelial monolayers.94 Physiology & Anatomy Functional activity; phagocytosis of outer segments Apical-basal polarization; distribution of channels, receptors, transporters; basal (VEGF, IP-10, RANTES, MCP-3) vs. apical (MCP-1, IL-6, IL-8, PEDF, TGF-beta1 & 2) secretion. Transepithelial resistance (tight junctions, esp. CLAUDIN 19) ~Ω/cm2 Resting membrane potential (apical: -50-60 mV). Fluid transport (5-10 µl/cm2h). Regulate volume and composition of subretinal space. Anatomy: cuboidal, confluent monolayer. Immunologic properties; T-cell suppression.  *As summarized by Barti et al.

Stem cells can be used for rescue therapy. In a preclinical model of glaucoma, for example, intravitreal somatic neural stem cells¹¹¹ and bone marrow-derived mesenchymal stem cells¹¹² can substantially reduce retinal ganglion cell death. In rhodopsin knockout mice, bone marrow-derived mesenchymal stem cells rescue photoreceptors.¹¹³ Subretinal bone marrow-derived mesenchymal stem cells rescue photoreceptors in RCS rats.¹¹⁴ hESC-derived RPE elaborate neurotrophic substances that have been shown to support photoreceptor survival in preclinical models of retinal degenerative disease.¹¹⁵

Although consideration of iPSCs as screening tools is beyond the scope of this brief review, one might note that iPSCs harbor the disease-associated genes of the patient from which they are derived. Cultures of these cells might be used for high-throughput screening to identify small molecules that reverse or halt the biochemical abnormalities induced by the disease-causing mutation(s). In this sense, the iPSCs can serve as a basis for developing pharmacological treatments for degenerative retinal diseases.

Diseases in which RPE cells appear to be targeted primarily include Best disease^116,117 and some forms of RP,^118,119 and secondarily include Stargardt macular dystrophy^120,121 and AMD.^40,122 RPE cell transplants are an attractive starting point for cell-based combination replacement and rescue therapy in the eye because hESCs and iPSCs can be induced to differentiate into RPE relatively easily, and one can generate large quantities of cells with stable genotype and appropriate phenotype. Currently, RPE differentiation from ESCs is a two-step process, typically requiring weeks in culture.^62,123,124 The first step involves converting ESCs or iPSCs into cells with neuroectodermal properties. The second step involves differentiating neuroectodermal cells into RPE cells. At this time, only a part of the ESC or iPSC culture is transformed into RPE, which may mean that the cultures are heterogeneous. hESC-derived RPE tends to resemble fetal RPE more closely than adult RPE, and iPSC-derived RPE seems to be in a unique differentiation state.^{62,68,125-127} Both hESC- and iPSC-derived RPE express differentiation markers: tryosinase (melanin); premelanosomal protein-17 (melanin); Bestrophin-1 (chloride channel); MERTK (phagocytosis); focal adhesion kinase; PEDF/VEGF (growth factors); and RPE65/RLBP1 (visual cycle).

In addition to the relative ease of producing differentiated RPE from stem cell progenitors, RPE cells integrate easily with host photoreceptors, and RPE cells elaborate trophic substances that support photoreceptors.^100,128,129 There is robust evidence for RPE transplant efficacy in pre-clinical models.⁹¹ However, in the case of AMD eyes, survival and proper differentiation on submacular Bruch’s membrane may be problematic.¹⁰⁰

Table 5. Results of Retinal Stem Cell Therapy in Preclinical Models  Disease Cell Type Delivery Route Effect  Rd1 & rd10 mouse130 BM-derived lineage-negative hematopoietic SCs Intravitreal Rescued photoreceptors (primarily cones)  Rhodopsin knockout mouse113 BM-derived MSCs Subretinal Rescued photoreceptors Ischemic retinopathy131 Endothelial progenitor cells Intravitreal Vascular repair & reversal of ischemic injury Rd1,132, mnd,133 & CRX-/-134 mouse Human ESCs Intravitreal Subretinal Rescued photoreceptors RPE 65-/- mouse145 ESC-derived RPE Subretinal Rescued photoreceptors* RCS rat114 BM-derived MSCs Intravitreal Rescued photoreceptors & preserved retinal function RCS rat62,64,99,125,135 Human ESC-derived RPE Subretinal Rescued photoreceptors & improved visual function RCS rat136 Human neural progenitor cells Subretinal Rescued photoreceptors & improved visual function RCS rat69 Human iPSC-derived RPE Subretinal Rescued photoreceptors & improved visual function RCS rat137 Human umbilical cord-derived SCs Subretinal Rescued photoreceptors & improved visual function Elov14 mouse125 Human ESC-derived RPE Subretinal Rescued photoreceptors Ush2a mouse138 Forebrain-derived progenitor cells Subretinal   SCs, stem cells BM, bone marrow MSCs, mesenchymal stem cells ESCs, embryonic stem cells iPSC, induced pluripotent stem cells *Teratomas formed in this study Reproduced with permission from Zarbin.1

Stem cell therapy has been effective in preclinical models of retinal degenerative disease, including models of RP and Stargardt macular dystrophy (See Table 5).

Stem cells are being used in human clinical trials to treat degenerative retinal diseases, including Stargardt macular dystrophy, AMD and RP (See Table 6). These studies represent early efforts in this area. Two of the studies have published preliminary results, and these studies are considered in greater detail below.

Figure. 3 Images of the human embryonic stem cell-retinal pigment epithelium transplantation site in a patient with Stargardt macular dystrophy. Color fundus photographs of the patient’s left macula preoperatively and postoperatively (A-C). The region inside the rectangle bisects the border of the surgical transplantation site and corresponds to macular atrophy not included in the surgical injection. A) Baseline macular color image with widespread retinal pigment epithelium and neurosensory macular atrophy. B) Color macular image one week after hESC-RPE transplantation. Note the mild pigmentation most evident in the region of baseline RPE atrophy. C) This pigmentation increased at week six. (D-G) Color fundus photographs and spectral domain ocular coherence tomography images at baseline (D) and month three after transplant (F). Color images show increasing pigmentation at the level of the RPE from baseline to month three. Registered SD-OCT images (E, G) show increasing pigmentation at the level of the RPE, normal monolayer RPE engraftment, and survival at month three (arrow) adjacent to the region of bare Bruch’s membrane devoid of native RPE. (Image reproduced with permission from Schwartz et al.141)

Stargardt Macular Dystrophy

Stargardt macular dystrophy is the most common macular dystrophy of childhood.¹⁴⁰ Currently, gene therapy (clinicaltrials.gov identifier: NCT01367444; sponsor: Oxford BioMedica) and nutritional supplementation (NCT01278277; sponsor: Catholic University of the Sacred Heart, Rome) also are under study for this condition. Experiments in an animal model of Stargardt disease indicate that hESC-derived RPE can rescue photoreceptors.¹²⁵ In one study, four months after subretinal transplantation of hESC-derived RPE into a patient with advanced Stargardt macular dystrophy, visual acuity improved from hand motions before surgery to 20/800.¹⁴¹ There was no improvement in the unoperated fellow eye. Pigmented cells at the transplant site seemed to proliferate during the four-month period of observation. Optical coherence tomography indicated that the pigmented cells were organized in a monolayer. OCT images of the retina overlying these cells did not demonstrate improved photoreceptor anatomy, and the subjacent choroid seemed unchanged also (See Figure 3). There was no evidence of teratoma formation or immune rejection of the transplanted cells. This patient is enrolled in a Phase I/II open-label, prospective, multicenter study to determine the safety and tolerability of subretinal transplantation of hESC-derived RPE cells in patients with Stargardt macular dystrophy and AMD. As part of the treatment protocol, the patient received a seven-week course of tacrolimus and mycophenolate mofetil starting one week before surgery. Per protocol, at week six after surgery, tacrolimus was discontinued and mycophenolate mofetil was continued for an additional six weeks. It is not clear whether the immune suppressive subretinal space⁹¹ will prevent rejection of these HLA Class II expressing cells after immune suppression is stopped.

AMD is the leading cause of blindness in persons older than 55 years in the United States.¹⁴² AMD can cause visual loss through two mechanisms: choroidal new vessels and geographic atrophy. Although there are effective treatments for CNVs,¹⁴³ there is no proven therapy for GA currently. A number of potential treatments are under study, and they are listed in Table 7.¹⁴³

One group has reported that four months after subretinal transplantation of hESC-derived RPE into a patient with GA, vision improved from 20/500 at entry to 20/200 by week two after surgery.¹⁴¹ Visual acuity was 20/320 by week six and remained stable at the three-month follow-up visit. Of note, mild visual improvement was also noted in the unoperated fellow eye after surgery. This patient received tacrolimus and mycophenolate mofetil as described above. 

In a prospective Phase I, nonrandomized open-label study of RP patients with best-corrected ETDRS visual acuity worse than 20/200,¹³⁹ three patients with RP and two with cone-rod dystrophy underwent intravitreal injection of autologous bone marrow-derived mononuclear cells with no adverse effects (and no documented benefit at 10-months follow-up).

Table 6. Human Stem Cell Trials for Retinal Disease*  Disease/Trial I.D.# Phase Cell Type Transplanted Center (PI) Sponsor Stargardt Macular Dystrophy (NCT01345006) I/II hES-RPE Jules Stein-UCLA (Schwartz) Wills Eye Hospital (Regillo) Moorfields Eye Hospital (Bainbridge) Advanced Cell Technology AMD-GA (NCT01344993) I/II hES-RPE Jules Stein-UCLA (Schwartz) Wills Eye Hospital (Regillo) Advanced Cell Technology AMD-GA (NCT01632527) I/II hES-RPE CHA Bundang Medical Center (Song) CHA Bio & Diostech Stargardt Macular Dystrophy (NCT01625559) I hES-RPE CHA Bundang Medical Center (Song) CHA Bio & Diostech AMD-GA (NCT01632527) I/II Human central nervous system SCs Retina Foundation of Southwest (Birch) Stem Cells Inc. AMD-GA (NCT01226628) I CNTO 2476 Wills Eye Hospital (Ho) Janssen Research & Development, LLC AMD-GA or CNV (NCT01518127) I/II Autologous bone-marrow derived SCs University of São Paulo, Brazil (Siqueira) University of São Paulo RP & cone-rod dystrophy (NCT01068561) I/II Autologous bone-marrow derived SCs University of São Paulo, Brazil (Siqueira et al.)139 University of São Paulo *Adapted from Zarbin.1 AMD, age-related macular degeneration GA, geographic atrophy CNV, choroidal neovascularization RP, retinitis pigmentosa hES-RPE, human embryonic stem cell-derived retinal pigment epithelium SCs, stem cells CNTO, human umbilical tissue-derived cells

In contrast to standard pharmacological monotherapy, transplanted cells can secrete numerous molecules that may exert a beneficial effect on the host retina and/or choroid even if they do not cure the underlying disease^{101,125,128,129,144} As noted previously, with a single transplant operation, many different pathways can be modified, which may reduce the chance of “escape” associated with monotherapy as well as the need for repeated drug administration. In addition, transplanted cells can replace dead cells (e.g., photoreceptors). Due to their pluripotency and unlimited proliferative capacity, stem cells seem to be the best starting material for cell-based therapy because these cells can be produced en masse safely, and they can be induced to differentiate into ocular cells with potential for replacement and rescue therapy. Preclinical studies demonstrate the feasibility of using ESCs and iPSCs for treating degenerative retinal diseases associated with abnormalities in the RPE and/or photoreceptors.

Some issues, however, may limit the use of stem cells in clinical practice, including: immunogenicity of the cells; stability of cell phenotype (both inherent and environment-induced); propensity of the cells to form tumors in situ; influence of the abnormal microenvironment that can accompany degenerative disease; and synaptic rewiring that accompanies retinal degeneration. In the case of non-exudative AMD, cell transplants might prevent GA progression (through replacement of dysfunctional or dead RPE) and might even bring about some visual improvement in selected cases (through rescue of dying photoreceptors). Cell-based therapy may one day be sight-restoring for patients who are blind due to retinal degenerations of various etiologies. RPE transplantation is an attractive starting point for this sort of therapy, since these cells can integrate with the host retina easily.  

Table 7. Potential Therapies for AMD in Clinical Trial  Compound Developer ClinicalTrials.gov i.d. Neurotrophic Factors  Ciliary neurotrophic factor Neurotech NCT00447954  Brimonidine Allergan NCT00658619 AL-8309B (tandospirone) Alcon NCT00890097   Visual Cycle Inhibitors Fenretinide ReVision NCT00429936 ACU-4429 Acucela NCT00942240 NCT01002950 Compliment Inhibitors Eculizamab Alexion NCT00935883 ARC1905 Ophthotec NCT00950638 FCFD4514S Roche NCT01229215 NCT00973011 Steroids Fluocinolone acetonide Alimera Sciences NCT00695318 Inhibitors of Amyloid-beta RN6G Pfizer NCT00877032 GSK933776 GlaxoSmithKline NCT01342926