RETINA AND RETINAL DISEASES
Retina is a transparent neural tissue for light reception within the eye. Mature retina is composed of 6 types of neurons, 1 type of epithelial cells, and 1 type of glial cells. All of these cells are derived from a single type of retinal progenitor cells, the neuroblast, in a sequential order. The 6 types of neurons are (from posterior to interior) the rod and cone photoreceptors, the amacrine cells, the horizontal cells, the bipolar cells, and the retinal ganglion cells. They form a characteristic laminar structure with 11 layers (from posterior to interior: pigment epithelium, outer segment and inner segment of photoreceptors, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane).1
Diseased retina would weaken the reception of the light and lead to vision loss or even blindness. This is mainly caused by the death of neurons or degeneration of the retinal pigment epithelium. Common retinal diseases include glaucoma, retinitis pigmentosa, diabetic retinopathy, and age-related macular degeneration (Table 1). Being the second leading cause of blindness in the world,2 glaucoma is a group of chronic, degenerative optic neuropathies, which are characterized by a slow progressive degeneration of retinal ganglion cells and their axons.3 This results in a distinct appearance of the optic disc, optic-nerve cupping, and a concomitant pattern of visual loss. Retinitis pigmentosa is characterized by a classic pattern of difficulties in dark adaptation and night blindness in adolescence, loss of midperipheral visual field in young adulthood, and central vision in later life. These are due to the severe attenuation of rod and cone photoreceptors.4 Retinitis pigmentosa is one of the hereditary degenerative diseases, affecting 1 in 4000 individuals. Diabetic retinopathy is the most common cause of vision impairment in working-age adults in developed countries. The retinal changes include the formation of retinal capillary microaneurysms, development of excessive vascular permeability, vascular occlusion, proliferation of new blood vessels and accompanying fibrous tissue on the surface of the retina and optic disc, and contraction of the fibrovascular proliferations and the vitreous.5 Age-related macular degeneration is the leading cause of irreversible blindness in people 50 years or older in the developed world.6 It influences the central portion of the retina (the macula).7 Early age-related macular degeneration is characterized by drusen (pale yellowish lesions) or by hyperpigmentation and hypopigmentation of retinal pigment epithelium in the macula. Late age-related macular degeneration is divided into “nonexudative” and “exudative” forms. Nonexudative form (geographic atrophy) starts with a sharply demarcated round or oval hypopigmented spot in which large choroidal vessels are visible, whereas exudative form, characterized by choroidal neovascularization, is the detachment of the neuroretina or retinal pigment epithelium from Bruch membrane by serous or hemorrhagic fluid.8 Traditional and current treatment regimens for the retinal diseases are based on surgical or medical interventions to slow down the disease progression and limit visual loss. However, the number of retinal cells would still diminish, and the diseases could not be completely cured. Therefore, new and effective therapies should be developed against these sight-threatening diseases. Recently, there is an emerging role of stem cells for retinal diseases.
TYPES AND SOURCES OF STEM CELLS
Stem cells are undifferentiated cells defined by their abilities to self-renew and differentiate into mature cells. Stem cells can be characterized according to their differentiation ability (potency) and the developmental stage (Table 2). For potency, the stem cells can be classified into toti-, pluri-, multi-, oligo-, or unipotent cells. Pluripotency refers to the ability of cells to differentiate into any cell type of the 3 germ layers (ectoderm, endoderm, and mesoderm), whereas multipotency refers to the ability of cells to differentiate only into a closely related family of cells.9 Pluripotent stem cells are the origin of all somatic and germ line cells in the developing embryo.
For developmental stage, stem cells can be embryonic or adult origin. Embryonic stem cells (ESCs) are derived from the inner cell mass of a blastocyst before gastrulation, and they are considered as the gold standard for pluripotent stem cells. Adult stem cells are the stem cells found in fully developed tissues, and they are thought to be tissue specific. The function of adult stem cells is the maintenance of adult tissue specificity by homeostatic cell replacement and tissue regeneration.10 The existence of adult stem cells has been reported in multiple organs including brain, heart, skin, intestine, testis, muscle, and blood, among others. Hematopoietic stem cells, mesenchymal stem cells (MSCs), periodontal ligament–derived stem cells, and spermatogonial stem cells are the conveniently accessible adult stem cell populations in the adult body. It is believed that adult stem cells are only able to differentiate into progeny cells of their tissues of origin. An increasing number of studies, however, report that adult stem cells are capable of giving rise to cells of an entirely distinct lineage.10 This suggests that pluripotent adult stem cells could also be present in the adult body.11–14 In addition, pluripotency can also be conferred to terminally differentiated adult cells by the technique of “induced pluripotent stem cells” (iPSC). In this process, adult skin fibroblasts were induced into a pluripotent state by the forced expression of key transcription factors (OCT4, SOX2, KLF4, and c-MYC)15 or (OCT4, SOX2, NANOG, and LIN28).16 Despite the low reprogramming efficiency, this has become a convenient method for generating new pluripotent stem cell lines for research from differentiated adult cells.
RETINAL RESEARCH ON STEM CELLS
Stem cell therapy on retinal diseases are based on 2 theories: cell replacement and neuroprotective effect. Cell replacement therapy hypothesizes that new retinal cells could be generated from stem cells so as to substitute the damaged cells in the diseased retina. In theory, photoreceptor loss in retinitis pigmentosa and retinal pigment epithelial loss in age-related macular degeneration can be rescued by cell replacement therapy. This leads to the establishment of various protocols on the induction of ESC into retinal lineage (Table 3).17–20 A common initial step for all protocols is the embryoid body formation (or cell aggregation) in the bacterial-grade dish or low adherent plate. The floating cells would be supplemented by 100 ng/mL Dkk-1 and 500 ng/mL Lefty-A for 5 to 9 days18,20 or 5 ng/mL IGF-1, 1 ng/mL Dkk-1, and 1 ng/mL noggin for 3 days.19 The neurospheres would then be transferred to the adherent culture surface (poly-D-lysine/laminin,17 poly-D-lysine/laminin/ fibronectin,18,20 or Matrigel/poly-D-lysine, Collaborative Research, Waltham, MA).19 The adhered cells would be cultured for 12 to 28 days with the supplement of ITSFn medium and 5 ng/mL bFGF17; 5% fetal calf serum and 100 ng/mL Activin-A18; 10 ng/mL noggin, 10 ng/mL Dkk-1, 10 ng/mL IGF-1 and 5 ng/mL bFGF19; or 10 μM γ-secretase inhibitor DAPT, 50 ng/mL aFGF, 10 ng/mL bFGF, 1 mM taurine, 3 nM Shh, and 500 nM retinoic acid.20 These retinal induction protocols could generate 82% to 86% Pax6+ cells and 0.01% to 36% rhodopsin+ cells. Similarly, iPSC can also be induced into retinal lineage.21
When human ESC-derived retinal committing cells are grafted into the retina of the animal models, these cells can survive in the retina.22–24 Moreover, subretinal grafting promotes maturation of these retinal committing cells into photoreceptor cells, whereas epiretinal grafting enhances the integration of these retinal committing cells into the retinal ganglion cell and inner nuclear layers.22 The integrated cells express markers of relative retinal layers and the synaptic marker,23 suggesting that the transplanted cells could communicate with the endogenous retinal cells. Furthermore, no teratomas formation was detected in the transplanted eyes,23 suggesting that the undifferentiated ESC might not be present within the induced retinal cells or they could be subjected to differentiation by the endogenous retinal niche. Importantly, these retinal committing cells can also restore the light response and the electrophysiologic function of the retina in the animal models resembling photoreceptor loss.23,24 In addition to photoreceptor cells, retinal pigment epithelial cells can also be manufactured from human ESC. The human ESC-derived retinal pigment epithelial cells perform the same as the natural retinal pigment epithelial cells so that they can replace the endogenous retinal pigment epithelial loss in the animal models, rescue the visual function, and sustain the photoreceptor integrity without any teratoma formation.25 Similar to ESC, iPSC-derived retinal committing cells can also restore the structure and function of the retina in the degenerative mice.26
Stem cell replacement therapy for diabetic retinopathy is different from that for retinitis pigmentosa and age-related macular degeneration. The major clinical manifestation of diabetic retinopathy is the leakage of retinal microvasculature (Table 1).5 This is due to a progressive dysfunction and death of endothelial cells, pericytes, and vascular smooth muscle cells in retinal vessels.27 Therefore, instead of replacing the retinal neurons, stem cell replacement therapy for diabetic retinopathy is aimed to repair the damaged retinal vascular system. Endothelial progenitor cells, which have the ability to home in on areas of vascular insufficiency and to participate in endothelial repair and neovascularization, have been proposed to be used in the clinical trial for diabetic retinopathy.28,29
Although adult stem cells, such as MSC, are able to be induced into retinal cells in vitro,30,31 they showed no evidence of differentiation into retinal cells or integration into retina after intravitreal grafting into the animal eyes.32 Nevertheless, intravitreal transplantation of MSC promotes photoreceptor cell survival in an animal model for retinitis pigmentosa33,34 and increase retinal ganglion cell survival and reduce retinal ganglion cell axon loss in an animal model for glaucoma.35,36 Mesenchymal stem cells are mainly situated in the vitreous cavity,35 and only some of them migrate into the host retina.33,36 Similarly, intravenous injection of MSC improves blood-retinal barrier integrity and reduces leakage in an animal model for diabetic retinopathy.37 Because the transplanted MSC remains undifferentiated and there is no direct cell replacement, these studies suggested that MSC would exert “neuroprotective effect” by secreting trophic factors, such as vascular endothelial growth factor, basic fibroblast growth factors, insulin-like growth factor I, ciliary neurotrophic factor, brain-derived neurotrophic factor, glial cell line–derived neurotrophic factor, and hepatocyte growth factor.38 The trophic factors could enhance survival of neurons, reduce cell apoptosis, or activate endogenous cell growth. Moreover, MSC might also remodel the extracellular matrix or activate the endogenous cell by cell-cell interaction.39 In addition, MSC have strong immunomodulatory properties against alloreactivity of T lymphocytes and dendritic cells.40 All these paracrine actions could modulate the microenvironments and niches within the diseased retina so that the retinal neurons can be protected and repaired.
The preclinical animal studies, based on cell replacement therapy and neuroprotective effect, provide evidence of visual improvement by stem cell transplantation, which establishes a solid foundation for future clinical studies in human patients.
CLINICAL APPLICATIONS OF STEM CELLS ON RETINAL DISEASES
Retina is an accessible tissue, where cells to be transplanted can be delivered by intravitreal or subretinal injection to specific diseased area.41 The advantage of local delivery eliminates the disturbance to other organs. Moreover, eye is an immune privilege site,42 which reduces rejection of transplanted cells. In addition, in vivo imaging techniques, such as optical coherence tomography and electroretinography, can closely monitor the structure and function of the retina upon transplantation.43,44 These advantages facilitate the transition of preclinical studies to human clinical trials for retinal diseases. Currently, there are 4 ongoing human clinical trials using stem cell therapy on retinal diseases. Two of them are ESC-based replacement therapy, whereas the other 2 are adult stem cell–based neuroprotective therapy.
In 2011, Advanced Cell Technology (Santa Monica, Calif) launched 2 phase I/II clinical trials of ESC-derived retinal pigment epithelial cell replacement therapy for Stargardt macular dystrophy (http://clinicaltrials.gov/ct2/show/NCT01345006) and advanced nonexudative age-related macular degeneration (http://clinicaltrials.gov/ct2/show/NCT01344993).45 The human ESC-derived retinal pigment epithelial cells were manufactured with 99% purity and free of pathogens. Fifty thousand manufactured retinal pigment epithelial cells were delivered to a patient with Stargardt macular dystrophy and another patient with advanced nonexudative age-related macular degeneration by subretinal injection to the diseased macular region. The patients were under immunosuppression 1 week before and 12 weeks after the transplantation. There is no evidence of hyperproliferation, ectopic tissue formation, teratoma formation, apparent rejection, intraocular inflammation, or retinal detachment 4 months after transplantation. The operated eye of the patient with Stargardt macular dystrophy showed improvement from hand motions to visual acuity of 20/800, whereas the visual acuity of the treated eye of the patient with advanced nonexudative age-related macular degeneration improved from 21 to 28 ETDRS letters.
The first intravitreal injection of human adult stem cells into human subject (the initial feasibility study) was reported in 2008.46 One hundred eighty million autologous bone marrow–derived mononuclear cells and 0.5 million CD34+ cells were transplanted into a patient with diabetic retinopathy. The injected cells were located on the retinal surface in the inferior fundus periphery on postoperative day 1. Although the visual acuity was not improved, adverse effects, such as inflammation or infection, were not observed. This affirms the safety of transplantation of autologous adult stem cells into the human eye. The first clinical trial of human adult stem cells on retinal diseases is a phase I human clinical trial held by University of São Paulo (http://clinicaltrials.gov/ct2/show/NCT01068561).47 Ten million autologous bone marrow–derived mononuclear cells were injected intravitreally into 3 patients with retinitis pigmentosa and 2 patients with cone-rod dystrophy. No structural change or functional toxicity was observed 10 months after the transplantation. One-line improvement in best-corrected visual acuity was demonstrated in 4 of 5 patients as early as 1 week after the transplantation. Moreover, 2 patients showed responses for dark-adapted standard flash stimulus. Another clinical trial on adult stem cells was launched by Centocor, Inc, d/b/a Janssen Research and Development (Beerse, Belgium; http://clinicaltrials.gov/ct2/show/NCT01226628). This is a phase I/II clinical trial assessing the safety and clinical response of umbilical tissue–derived cells (CNTO 2476) to the temporal macula of patients with advanced nonexudative age-related macular degeneration through a microcatheter delivery system. The preliminary results of this study have not been reported yet.
These clinical trials do show a promise of hope as to the use of stem cell therapy to treat retinal diseases in the future. However, a larger cohort of patients is needed to confirm the repeatability and reproducibility of these stem cell therapies. In addition, long-term follow-up is also necessary to ensure the safety of transplantation and to monitor any adverse effects.
POTENTIAL CHALLENGES AND FUTURE PROSPECTS
The cell source for stem cell therapy on retinal diseases can be allogenic or autogenic. Human ESC comes from the inner cell mass of human blastocysts. Therefore, ESC used for therapy must be allogenic, which the transplanted donor cells do not originate from the recipient. This raises a concern about the immunogenic response of the host, and the need for immunosuppressive therapy concurrent with ESC transplantation.48 Differentiated adult cells used for the generation of iPSC can be collected from the recipient body, circumventing the problem of immune rejection. Moreover, transplantation of adult stem cells can be autogenic or allogenic. Immunosuppression is not necessary for MSC transplantation because MSCs have strong immunomodulatory properties against alloreactivity of T lymphocytes and dendritic cells.40,49 Similarly, MSC and periodontal ligament–derived stem cells can also inhibit the proliferation of peripheral blood mononuclear cells.50 Therefore, MSC is an ideal source for stem cell transplantation in which immunosuppression is minimized.
Induced pluripotent stem cells can avoid the use of ESCs, which has been hampered by the moral, legal, and ethical dilemma surrounding the use of human embryos for derivation of the stem cell lines.51 However, there are technical hurdles concerning generation of iPSC.52 First, the delivery of reprogramming factors (OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC) relies on the use of viral vectors for delivery.15 Retroviral sequences could integrate into the DNA of the host cells, potentially disrupting the gene structure and resulting in an aberrant phenotypic expression. Ultimately, this could result in pathologic mutations and cancer formation. Alternative methods such as direct protein or small molecule delivery have been adopted, but the reprogramming efficiency of these techniques is lower than with viral vectors.53,54 Second, 2 of the reprogramming factors, c-MYC and KLF4, are protooncogenes, further raising the concern of cancer formation. Omitting c-MYC would lower the reprogramming efficiency, whereas silencing c-MYC could lead to its reactivation. Moreover, reprogramming can induce other genomic changes, such as DNA mutations,55 copy number variations,56 and chromosomal aberrations.57 Genomic instability could have unpredictable and undesirable effects on the reprogrammed cells. Furthermore, iPSC carry their epigenetic signatures from the original differentiated adult cells.58 The reprogrammed cells, unlike ESC, may not develop into some cell types. In contrast, the pluripotent status of some adult stem cells is naturally acquired and does not require reprogramming by the introduction of pluripotent transcriptional factors, thus eliminating the use of viral vectors and the chance of aberrant chromosomal changes. Pluripotent adult stem cells can easily be isolated and purified by cell surface markers such as CD49f, SSEA-3, and SSEA4.11,13,59,60
Embryonic stem cells and iPSC have the potential to form teratomas in the host. Tumorigenic potential can be reduced by differentiating ESC into lineage-specific progenitor cells or mature tissue cells before transplantation.45 Tumorigenic potential remains a concern if the entirety of the ESC population does not completely differentiate into fully mature cells, although a recent study suggests that the teratoma-forming cells could be removed by the antibody against SSEA-5.61 On the contrary, the pluripotent adult stem cells (hematopoietic stem cells, MSC, and periodontal ligament–derived stem cells, except spermatogonial stem cells) do not form teratomas in vivo, eliminating the need for preemptive differentiation of pluripotent adult stem cells into mature specialized cells.
Before any human clinical trial studies, stem cells or their derived cells need to be tested by a Good Manufacturing Practice–compliant study in National Institutes of Health III immune-deficient mouse model, recommended by the Food and Drug Administration.25,29 Long-term data (spanning the life of the animals) is needed to provide the evidence of no teratoma formation. However, because stem cell products become more widespread and regulated under various conditions, there is an increasing need for global standardization and regulation of the processes to ensure viable application of these products in a clinical setting. The Food and Drug Administration regulates interstate commerce in human cells and tissue-based products under the Public Health Service Act and the Code of Federal Regulations for Food and Drugs.62 Human cells and tissue-based products are defined as “articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient.”62 Human cells and tissue-based products must be (1) minimally manipulated, (2) intended only for homologous use, and (3) not combined with another article (except for water, or sterilization, preservation, or storage agents), and (4) either they (a) have no systemic or metabolic effect or (b) be for autologous use, allogeneic use in first- or second-degree blood relative, or reproductive use. Stem cells fall under the criteria for human cells and tissue-based products as stated by the Food and Drug Administration. Unlike iPSC, pluripotent adult stem cells can be minimally manipulated because their pluripotent state occurs naturally. Unlike ESC, pluripotent adult stem cells are suited for autologous use. Similar to ESC and iPSC, pluripotent adult stem cells are able to differentiate into specialized cells of the 3 germ layers.11–14,59,60
Beside the choice of stem cells, the delivery of stem cells also warrants attention. Stem cells or their derived cells can be transplanted by intravitreal, subretinal, or intravenous injection.41,63 The choice of injection mode depends on the types of diseases. For retinitis pigmentosa and age-related macular degeneration, subretinal injection is preferred because these diseases affect the photoreceptors and retinal pigment epithelium. For glaucoma and diabetic retinopathy, intravitreal injection is preferred because the superficial layers of cells are affected. Intravenous injection of stem cells is not recommended because stem cells would be attracted by other organs such as the lungs64 and less likely to travel to the eye. Moreover, the delivery of stem cells can be in form of cell suspension or a cell sheet.63 The immune privilege is lower for cell suspension than as an intact cell sheet.65 Therefore, biomimetic scaffolds will be a future research direction to manipulate the delivery of stem cells into the correct diseased location of the retina.66,67
Current retinal induction protocols were only tested on ESC and iPSC.17–21 Because pluripotent adult stem cells are recently discovered,11–14,59,60 the retinal induction protocols should also be tested on these conveniently accessible stem cells. The validation of retinal induction potential of pluripotent adult stem cells will revolutionize the stem cell therapy in the field of retinal research. Nevertheless, the optimal stem cell types, the optimal cell number, the optimal cell stages, and the disease stages for transplantation are needed to be determined in future studies.
Stem cell clinical trials have advanced rapidly for a broad spectrum of diseases such as diabetes, neurodegeneration, immune diseases, heart diseases, and bone diseases.68 We are now approaching to the era of stem cell therapy for retinal diseases. Stem cells can be conveniently obtained from different sources: ESC, iPSC, and adult stem cells, and they can be guided to differentiate into different retinal cells by the retinal induction protocols.17–20 The manufactured stem cells and their derived cells are able to rescue the diseased retina in different retinal disease animal models by cell replacement therapy or neuroprotective effect.22–26,29,32–37 Moreover, preclinical Good Manufacturing Practice–compliant studies can ensure that stem cells and the derived cells are safe and would not form teratoma in the immune-deficient mice.25,29 In addition, there are ongoing clinical trials of ESC-derived retinal pigment epithelial cells, bone marrow–derived mononuclear cells, and umbilical tissue–derived cells on different retinal diseases.45,47 They are the stepping stone toward the application of stem cell products in human subjects. It is believed that stem cell therapy will eventually become the treatment of choice in regenerative medicine, especially with the use of adult stem cells.
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“It s not what you look at that matters, it’s what you see.”
- Henry David Thoreau