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.
Definitions and Classes
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.
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
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 vectors46-49 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).
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.
Replacement vs. Rescue Therapy
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
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.
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 disease116,117 and some forms of RP,118,119 and secondarily include Stargardt macular dystrophy120,121 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
Retinal Degenerative Disease
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.
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).
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 disease101,125,128,129,144 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.
Supported in part by Research to Prevent Blindness Inc. and the Joseph DiSepio AMD Research Fund. Contact Dr. Zarbin at the Institute of Ophthalmology and Visual Science, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Room 6155, Doctors Office Center, 90 Bergen Street, Newark, N.J. 07103. Phone: 973-972-2038; fax: 973-972-2068; email: email@example.com.
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