Cell-Based Therapies for Ocular Disease

Abstract

Cell therapy for ocular disease has made significant progress within the last decade. Stem and progenitor populations for many ocular cell types have been identified, and their behavior is now understood well enough to enable clinical application. Corneal epithelial progenitor cell therapy has benefited many patients and is now transitioning from a research technique to established clinical therapy. The application of embryonic stem cell-based therapy is in clinical development for Stargardt's macular dystrophy and dry age-related macular degeneration. These advances have been made possible, in part, by the inherent advantages of the eye as a place to develop and apply cell therapies and the foundation built on transplantation studies. Despite these advances, there are still areas of high unmet need that could benefit from cell therapy when further research identifies methods to identify, generate, and manipulate the progenitor populations. This review discusses, in practical terms, the application of cell therapies to the eye, progress that has been made and progress which remains to be made in the application of cell therapy to ocular disease.

Introduction

Cell therapies for diseases and conditions of the eye are advancing rapidly. Hundreds of patients have been treated in the research setting, and formal regulatory approval for cell therapies in the eye and broad availability to patients is within sight. The rapid advance of cell therapies in ocular disease is due to a number of factors that make the eye an ideal setting for the implementation of cell-based therapies (Table 1). The ability to surgically place the transplanted donor cells, under microscopic visualization if necessary, at the site of a lesion means that the donor cells do not have to migrate far to “home” to the site of action. This contributes to the efficiency of the transplant and reduces the number of donor cells needed. Restoration of the retina in diseases with widespread retinal degeneration is more challenging, but significant visual benefits can be gained by improving function in fairly small areas of the retina for these patients. These implantation procedures are generally much less invasive than those needed to place cell therapies into other areas of the central nervous system (CNS) or in specific sites in internal organs. Once placed at the desired site, transplanted cells can be followed by a variety of powerful imaging techniques, including OCT. Cells in the eye enjoy a partial immune privilege, and the issues of tissue matching, rejection, and immune suppression are significantly reduced.

Table 1.

Advantages of Cell Therapy in the Eye

Key advantage	Examples
Small number of cells needed	In corneal limbal stem cell transplant, as few as 3,000 cells can lead to clinical success¹⁰
Ability to transplant to precise locations under direct visualization	Implantation of RPE cells into the retina can be done under direct visualization of both the implantation site and the retinal atrophic area, and cells can be placed in the desired tissue layer²²
Noninvasive imaging to monitor cell fate	The site of implantation can be imaged repeatedly²²
Barriers to migration of transplanted cells outside the globe	Cell transplants into the eye have never been documented to leave it
Partial immune privilege	Allogeneic transplantation of ocular tissue without chronic immunosuppression is well established³¹
Teratomas expected to be easily seen and removed, less harmful to patients	Small tumors within the eye are easily seen and removed surgically
Easily accessible tissue without major surgery	Wounds and trauma created by ocular surgery are small and overall surgical risks are low

RPE, retinal pigment epithelium.

The eye also has a safety advantage for the development of cellular therapies. The dominant safety concern in the development of stem cell-based therapies is the development of teratomas or other abnormal growths.¹ In many tissues such as the brain and the spinal cord, even a small teratoma can have significant adverse consequences for the patient, and the potential for the donor cells to migrate to distant locations and form teratomas is worrying. This is not merely a theoretical concern, as at least one case of brain tumor secondary to stem cell transplantation has been reported.² In the eye, teratoma or growth formation can be monitored by existing imaging technologies, and the removal of a growth from an eye with poor vision is much less impactful to the patient than brain surgery. When considering cell therapy applied to the retina and interior of the globe, the eye is a naturally limited space that presents a barrier to cells escaping and causing complications elsewhere. These are among the reasons that cell therapies for ocular disease have made and continue to make great progress.

Corneal Diseases

Corneal epithelial cells

Corneal transplantation in the presence of an intact limbus results in the host corneal epithelial cells replacing the donor cells and repopulating the outer surface of the donor cornea.³ Conjunctival limbal autografts provided convincing evidence that the host epithelial cells repopulating the donor cornea originate in the corneal limbus.⁴ Further work has established that there is a stem cell niche in the limbus of the cornea that provides epithelial cells which can repopulate a damaged cornea and will replace the donor epithelial cells in a transplanted cornea. For patients with limbal stem cell deficiencies, allogeneic transplantation of a portion of the cornea and a portion of the limbus as a unit can be successful.⁵ Success rates are higher for human leukocyte antigen-matched grafts, but even nonmatched grafts can achieve clear central corneas at 2 years with a 70% success rate when done in conjunction with mitomycin C, amniotic membrane, and continuous systemic immunosuppression.⁶

The methods for explanting and expanding corneal limbal epithelial stem cells were first demonstrated by Pellegrini et al.⁷ in 1997, and now, several methods for the culture and transplantation of corneal limbal stem cells are well established in a number of laboratories and clinical centers (for review see Tseng et al.⁸). A small sample of the corneal limbus can be surgically harvested from the fellow eye or a donor eye without damaging the donor eye or compromising sight. This sample contains the limbal epithelial niche, and these cells can be expanded in culture and then transplanted to the recipient cornea, alone or with a biological matrix. Limbal stem cell transplantation can be very successful in patients with chemical burns to the cornea of one eye^9,10 (therefore having a supply of limbal stem cells for culture from the other eye), and success rates in this population are 70%–80%.

Both autologous and allogeneic transplantation modes have been explored. Patients who are candidates for allogeneic transplantation include those with bilateral damage or disease or limbal stem cell deficiency (LSCD). In a series of LSCD patients given allogeneic transplants from adult living-related donors where xeno-free culture conditions were used for expansion, more than 70% of eyes remained epithelialized long term.¹¹ Graft failure was often by conjunctivalization with grafted cells surviving in the cornea,¹² and conjunctivalization has been observed in failure of allogeneic grafts in the second decade postgraft.¹³ Based on these data, the success rate for allogeneic grafting is similar to autologous grafts.

While this technique has been very successful, there are still hurdles to be overcome. Restoration of normal vision depends on the properties of the corneal stroma, and patients often need additional treatments to correct this. The corneal stroma has its own population of stem cells, and application of these cells is advancing but is not yet in human clinical investigation (for review, see Pinnamaneni and Funderburgh¹⁴ and see section on corneal stroma cells). Limbal stem cell transplantation works best in patients who have had monocular acid or alkali burns (e.g., industrial accidents). It is less successful in patients with corneal epithelial defects due to Stevens–Johnson Syndrome or congenital connective tissue disorders.⁹

Corneal endothelial cells

Unlike the corneal epithelial cells, corneal endothelial cells do not appear to have a niche for stem cells in the adult and do not effectively repopulate transplanted corneas. Endothelial cells decrease in number with age and are not thought to divide or repopulate in normal adults,¹⁵ and this process is seen in transplanted corneas, limiting long-term survival.¹⁶ Disorders of the endothelial cell layer are important drivers of posterior corneal dystrophies such as Fuchs dystrophy. Autologous transplantation of corneal endothelial cells via endothelial keratoplasty is now a standard of care for severe endothelial dysfunction. The transplanted cells retain a limited ability to migrate and expand in vivo. Visual outcomes remain suboptimal with recent long-term follow-up analyses, indicating that only about half of patients reach 20/20 over 3 years.¹⁷ Due to the chronic shortage of donor corneas, generation of stem or progenitor cells that could replace the corneal endothelial layer is a significant unmet need. Methods for the harvest of pure populations of mature endothelial cells and short-term culture have recently been established.¹⁸ There is some evidence that cells of the trabecular meshwork (TM) may serve as a progenitor population for endothelial cells,¹⁹ but transplantation of this population to effectively repopulate the endothelium has not been demonstrated. In addition, there is no existing protocol for the differentiation of embryonic stem (ES) or induced pluripotent stem (iPS) cells into corneal endothelium nor identification of unique cellular markers for putative endothelial progenitor cells. This is an area of active research in several laboratories.

Corneal stroma

Disorders of the corneal stroma are relatively common, and involve degeneration of the collagen matrix with weakening of the corneal structure leading to keratoconus. After transplantation of the cornea, host stromal cells can be found in the graft tissue, suggesting that there is a population of stromal cells which are capable of migration and repopulation of the graft.²⁰ Stem cells can be found in the stroma (for review Pinnamaneni and Funderburgh¹⁴), and progress is being made in the differentiation of ES cells into stromal keratocytes.²¹

Age-Related Macular Degeneration and Retinal Dystrophies

There are a number of cell therapies for retinal disease in clinical and preclinical development, and the first-ever human embryonic stem (hES) cell-derived therapy is currently in clinical trials for retinal disease²² (Table 2 contains a summary of cell therapies in development). Genetic retinal dystrophies, including retinitis pigmentosa (RP) and Stargardts disease, are attractive targets for cell therapy, as these diseases involve cell autonomous degeneration of the photoreceptors driven by defined genetic mutations in proteins critical to and, in some cases, unique to photoreceptor function, or in the case of some forms of RP, to defects within the retinal pigment epithelium (RPE) cells. There is a high level of unmet need, as these patients go blind at a young age and there is no cure or palliative therapy available.

Table 2.

Cell Therapy Clinical Trials in Ophthalmology

Disease	NCT	Cell type	Sponsor
Bechet's disease	NCT00550498	Adult BM MSCs–autologous	Tehran
Retinitis pigmentosa	NCT01068561	Adult BM MSCs–autologous	University of Sao Paulo
	NCT01560715	Adult BM MSCs–autologous	University of Sao Paulo
	NCT01531348	Adult BM MSCs–autologous	Mahidol University, Thailand
AMD (wet or dry)	NCT01518127	Adult BM MSCs–autologous	University of Sao Paolo
Geographic atrophy	NCT01226628	Umbilical (CNTO 2476)	Centocor (J&J)
	NCT01344993	MA-09 hES cells differentiated into RPE, suspension	Advanced Cell Therapy
	NCT01632527	hNSCs	Stem Cells, Inc.
Stargardt's disease	NCT01345006	MA-09 hES cells differentiated into RPE, suspension	Advanced Cell Therapy
	NCT01469832	MA-09 hES cells differentiated into RPE, suspension	Advanced Cell Therapy
	NCT01625559	MA-09 hES cells differentiated into RPE, suspension	CHA Bio & Diostech Co., LTD (Korea)
Corneal surface repair	NCT01489501	Oral mucosal epithelial cell sheets	CellSeed France SARL/FGK Clinical Research GmbH
	JPRN- UMIN000006745	Cultivated autologous oral mucosal epithelial cell sheet	Dept. Ophth, Tohoku Univ. Grad School of Medicine (Japan)
	JPRN- UMIN000005400	Cultivated autologous oral mucosal epithelial cell sheet	Dept Ophth, Osaka University Graduate School of Medicine
	NCT00491959	Cultivated autologous oral mucosal epithelial cell sheet	National Taiwan University Hospital
	NCT00845117	Limbal stem cells, autologous or allogeneic	University Hospital, Antwerp
	NCT01562002	BMSCs on amniotic membranes	Instit. Univ. De Oftalmobiologia Aplicada, Valladolid, Spain
	NCT01619189	Limbal stem cells, autologous or allogeneic	Centre Hospitalier d'Ophth. des Quinze-Vingts, Paris
	NCT00736307	Limbal stem cells, autologous or allogeneic	Royan Institute, Tehran
	NCT01470573	Limbal stem cells, autologous	Fundacion Clinic per a la Recerca Biomedica (Spain)
	NCT01123044	Limbal stem cells, autologous	Ministry of Health, Malaysia
	NCT01237600	Limbal stem cells, autologous	Ministry of Health, Malaysia
Optic nerve atrophy	ChiCTR-TNRC-11 001491	Unknown	General Hosp of the Chinese People's Armed Police Force (China)
Ischemic retinopathy	NCT01518842	BMSCs	University of Sao Paulo

hES, human embryonic stem; BMSC, bone marrow stem cell; MSC, mesenchymal stem cell; hNSC, human neural stem cell; AMD, age-related macular degeneration.

There are 3 general approaches to cell therapy being explored at present. The first and most advanced is the transplantation of differentiated RPE derived from ES cells into the subretinal space. The second is the transplantation of retinal, neural, or other cells into the eye such that they provide primarily trophic support to the host cells. The third is the transplantation of partially differentiated retinal progenitor cells derived from multipotent progenitor populations that will integrate into the host retina and form mature retinal elements, including photoreceptors, in situ.

Lessons from transplantation

Transplantation of the retina has been explored in both animals and humans in several forms, including the transplantation of the neural retina as an intact sheet with attached RPE, macular translocation, and transplantation of the choroid and RPE without the associated neural retina. There are important differences in the outcome of these studies in various diseases, and they provide insights into the potential for stem and progenitor cell therapy.

RPE Transplantation and Macular Translocation

The objective of RPE transplantation and macular translocation is to position healthy RPE under the diseased neural retina and thereby restore retinal function. Macular translocation is an autologous transplant in which the central portion of the neural retina is separated from the underlying RPE, rotated around the optic nerve, and reattached to the RPE. In age-related macular degeneration (AMD) patients with localized RPE dysfunction under the fovea, this positions the fovea over healthy RPE. In a second surgery, the entire eye is then rotated in the reverse direction to restore appropriate visual alignment. In RPE transplantation, a section of healthy RPE is taken from the peripheral retina and implanted underneath the fovea to replace the diseased RPE.

Macular translocation in retinal choroidal neovascularization (CNV) produces improved visual acuity, with some patients gaining 3 or more lines (15 letters or more) of best-corrected visual acuity (BCVA) that is persistent for at least 5 years.^23,24 Transplantation of RPE/choroid in CNV patients can also have a positive outcome that is persistent, although the proportion of patients experiencing significant gains in BCVA is lower.²⁵ Transplantation of the RPE sheet alone using gelatin to support the RPE sheet has been reported without visual improvement.^26,27 CNV generally does not recur although there is one documented case.²⁸ This is compelling evidence that replacement of the dysfunctional RPE and choroid can have a beneficial effect in patients with CNV. The picture is less clear in geographic atrophy (GA). After macular translocation, recurrence of RPE dysfunction under the translocated fovea has been seen within 9 months postoperatively.²⁹ However, no recurrence was reported in long-term follow up of GA patients with transplants of RPE choroid.³⁰ These results argue that in both forms of AMD, transplantation of RPE has therapeutic potential. Both macular translocation and RPE-choroid transplantation represent intensive surgical procedures with high rates of complications, making them unsuitable for wide application as therapies.

Transplantation of Neural Retina

Transplantation of the fetal neural retina as a sheet has been extensively investigated³¹ and has shown positive effects in animals³² and humans³³ when transplanted with the RPE. Recovery of vision is modest as measured by VA, although all the patients in which this procedure has been tried had very poor vision before surgery. The lesson from such full-thickness retinal transplantation is that long-term survival of fetal retinal sheets transplanted into the adult can be achieved without tissue matching or immunosuppression. This suggests that allogeneic cell therapy can be done without rejection issues. Culture of whole retinas from embryonic or fetal animals followed by transplantation has been investigated in many models. It is important to note that while improvements can be seen, the relative contribution of the release of trophic factors versus integration and function by the transplanted cells is not well defined.

Retinal pigment epithelium

Both ES and iPS cells can be differentiated into RPE efficiently.^34,35 The differentiated cells will assume an RPE phenotype, including the formation of a polarized epithelium that is capable of phagocytosis of photoreceptor outer segments. More recently, it has been found that there is an RPE stem cell population in the adult human retina which can be expanded efficiently and differentiated into mature RPE.³⁶ These RPE stem cells (RPESCs) can also form cells of mesenchymal lineage, indicating their potential value in modeling types of RPE metaplastic disease. In development, the RPE has the potential to produce neural retina as well as RPE.³⁷ The transdifferentiation of RPE cells into retinal neurons can be accomplished in vitro using several different strategies.^38–40 RPE transplantation, thus, has the potential for regeneration of the neural retina as well as the RPE layer; however, the keys to leveraging this for therapy have yet to be elucidated. To date, cell therapy using RPE is aimed at providing functional RPE and not at replacing neural retina from the transplanted cells.

Application of RPE replacement therapy might be expected to be effective in both AMD (based on the transplantation data described earlier) and in retinal dystrophies such as RP and Stargardts disease. While most RP patients have mutations in genes expressed in the photoreceptors, a subset has mutations in genes that are important for RPE function such as the Mertk gene, a protein which is important for the phagocytosis by the RPE of photoreceptor outer segments. The rationale for transplantation is 2-fold—the transplanted RPE would replace dysfunctional RPE, and the powerful trophic effect that RPE exerts on photoreceptors can delay cell degeneration. RPE cells secrete a number of growth factors that are important for photoreceptor health and survival, including pigment epithelium derived factor,⁴¹ glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and even dopamine⁴² and the integration and phagocytic function of donor RPE may not be necessary to achieve beneficial effects. Provision of growth factors to the retina via implantation of transformed immortalized (non-stem cell) RPE cells has shown good results in animal models and is now in advanced clinical trials.⁴³

In rodent models of retinal dystrophy, transplantation of RPE results in structural and functional benefits. The most commonly used model is the Royal College of Surgeons (RCS) rat, which has a naturally occurring mutation in the Mertk gene. In this animal model, transplantation of allogeneic or xenogeneic RPE,⁴⁴ including iPS-derived⁴⁵ RPE into the retina, results in integration of the transplanted cells, preservation of pre and postsynaptic structures,⁴⁶ and improvements in cortical responses to visual stimuli^47,48 that are persistent.⁴⁹ It is important to note that the RCS rat model is relatively permissive and may, therefore, not be a clear indicator of efficacy in human disease. However, RPE transplantation is also effective in the Elov14 mouse model of Stargardts disease,⁵⁰ and in the RPE65 mouse model of RP,⁵¹ increasing confidence in the approach.

Transplantation of RPE can be done by injecting a suspension of RPE cells into a subretinal bleb or by subretinal implantation of a synthetic matrix containing cultured RPE. Implantation of a sheet is more surgically complex but offers the ability to know exactly where the implanted cells are and to restore the normal anatomical relationship between the RPE layer and the photoreceptors. In addition, the RPE cells can be transplanted as an adherent, polarized monolayer in a terminally differentiated state. Several different sheets have been tested in animals,^52,53 but none have reached human trials to date. In both of these procedures, the transplanted cells can be targeted to a specific site in the retina, but the coverage of the retina will be relatively small and any improvements in vision outside the transplanted area will depend either on cells migrating or on a trophic effect via diffusible factors.

In humans, 2 RPE cell suspension transplantation experiments have been completed. Transplantation of an RPE cell suspension has been compared with RPE-choroid transplantation in a small, randomized clinical study in neovascular AMD patients.⁵⁴ Both groups showed similar results, including limited, if any, vision gain. This result should be interpreted with caution, however, as all these patients had large lesions and poor vision at entry and had either failed or were unsuitable for all other therapies, including anti-vascular endothelial growth factor. In a separate report, transplantation of a suspension of fetal RPE did not result in visual improvements⁵⁵ in a single patient.

Two ongoing trials (see Table 2) are currently under way for testing the transplantation of hES cells differentiated into RPE into patients with Stargardts disease and dry AMD/GA.²² These trials are enrolling end-stage patients and are primarily meant to assess the safety and tolerability of the hES cell treatments. In these trials, hES cells derived from the blastocyst were differentiated into RPE and were >99% RPE at the time of implantation as judged by expression of the RPE markers bestrophin, MITF, and ZO-1. The cells were also found to express the neurectodermal marker Pax-6. Mature, fully differentiated RPE do not express Pax-6, suggesting that these cells were not fully differentiated at the time of implantation. RPE cells were implanted as a suspension into a retinal bleb at a dose of 50,000 cells in 150 μL. The blebs were placed in the pericentral macula in an area that “was not completely lost to disease.” Patients were immunosuppressed for 12 weeks post-transplant. The results of 2 patients at 12 weeks post-transplant have been reported, and no aberrant growth or hyperproliferation has been detected. In addition, both patients did not lose vision, although both patients had very poor vision at entry, which limits the ability to detect visual loss or gain. The sponsor of this trial, Advanced Cell Therapy, announced in October 2012 that they had received regulatory approval for dose escalation to 100,000 cells per injection and plan to progress to 200,000 cells per injection in the highest dose cohort (www.advancedcell.com/news-and-media/press-releases/act-announces-approval-to-treat-additional-stargardtandrsquo-s-disease-patients-with-higher-rpe-dosage-in-both-us-and-european-clinical-trials/index.asp).

Retinal neural elements

While RPE transplantation may be beneficial in some diseases, the ultimate goal of cell therapy for most retinal diseases is the ability to replace the neural elements of the retina. In lower animals, full retinal regeneration is possible. In mammals, all of the retina, including RPE, neurons, and support cells, can arise from a single progenitor in development.³⁷ Retinal progenitors maintain the ability to differentiate into diverse neuronal and glial cell types late into development in the rat.⁵⁶

Many source cells for the production of retinal elements have been investigated.⁵⁷ For therapy of glaucoma, replacement of the retinal ganglion cells (RGCs) is the target (see Glaucoma section); while in RP and GA, replacement of the photoreceptor cells is the key challenge. Photoreceptors are a unique cell type that can be placed surgically into their desired location but need to express a very specific phenotype which is seen nowhere else.

As a general rule, neural precursors, including CNS neural progenitors and pluripotent cells that have been differentiated into neural progenitors, integrate into the retina and develop neural or glial phenotypes. However, these cells do not produce fully differentiated photoreceptors and, to date, only retinal progenitors or pluripotent cells specifically differentiated toward photoreceptors can become photoreceptors. This is thought to reflect a lineage restriction that is specific to the optic cup precursors during development well before the invagination of the optic cup. hES cells self-organize into optic cup structures containing differentiated photoreceptor cells in vitro,⁵⁸ but the conditions to accomplish this in transplants have not yet been defined.

Progenitor Cells

A highly investigated source cell for the production of retinal elements are retinal progenitors from embryos or fetal animals. These cells have already moved beyond the RPE/neural retina decision and are committed to the neural retina fate. Retinal progenitors can be harvested from mice at postnatal day 1, and these retinal progenitors when transplanted can migrate to the photoreceptor cell layer and differentiate into what appear to be interneurons rather than photoreceptors.⁵⁹ The transplanted cells rescue host photoreceptors and preserve light sensitivity as measured by suppression of wheel running in lighted conditions, but differentiation of the transplanted cells into photoreceptors is rare. Retinal progenitors can be isolated via similar procedures from similar stages of development in rats (E17), and expression of rhodopsin in cells integrating into the outer nuclear layer has been documented.⁶⁰ Retinal progenitors allotransplanted into the P347L transgenic pig model of RP not only express photoreceptor markers but also assume rosettes that are reminiscent of normal photoreceptor development^61,62 although the preservation of visual function has not been demonstrated. Given the strong effect that transplanted cells can have on host retina, it is yet unclear whether the beneficial effects on visual function reflect functional photoreceptors formed by donor cells, even though rhodopsin may be expressed. However, it is encouraging that the integration of transplanted cells into the retina and improvements in vision are seen across multiple retinal degeneration models and across multiple species, including models which are highly relevant to human disease such as the transgenic RP models.

A second approach has been to transplant neural rather than retinal progenitors. Neural progenitor cells are better understood than retinal progenitors, and several groups, including StemCells Inc and ReNeuron, are studying these cells clinically. Stem Cells Inc recently started enrolling a study of their human neural precursor cell line in dry AMD patients (http://investor.stemcellsinc.com/phoenix.zhtml?c=86230&p=irol-newsArticle&ID=1741717&highlight=). Transplantation of human forebrain-derived neural progenitors into the retina in a mouse model of usher syndrome (Ush2a mice) decreased photoreceptor degeneration and improved visual acuity measured using optokinetic testing.⁶³ In this case, the transplanted cells remain in the subretinal space. In the RCS rat model, retinal transplantation of hNPC^ctx human cortical progenitors at postnatal day 21 results in improved visual acuity using optokinetic testing at postnatal day 100⁶⁴ and preserved b-wave by ERG. Interestingly, in this experiment, there was no difference in optokinetic performance between animals transplanted with hNPC^ctx and those transplanted with a line of hNPC^ctx expressing GDNF, although there was a difference in b-wave favoring the GDNF-expressing transplants. The optokinetic effect persists at least to day 150, a point where the control animals have no detectable optokinetic responses.⁶⁵ These animals also show dramatically improved luminance thresholds. Histopathological analysis shows that transplanted human CNS progenitors can migrate from the implantation site into the host retina as well as in the subretinal space, and that photoreceptors including cones are preserved in the retina.⁶⁶ The ability of transplanted CNS progenitors to migrate into the retina after subretinal transplantation has also been shown in the cat.⁶⁷

An important barrier to the clinical use of retinal progenitor cells derived from embryonic animal or human retina has been the difficulty in expanding these cultures. These cells tend to lose their ability to form neurons on repeated passage.⁶⁸ Retinal progenitor cells taken from laboratory animals can be pooled, diminishing the need for expansion over many passages. In human studies, retinal progenitor cells for clinical use are taken from a single embryo donor and expanding these cultures sufficiently to treat a large number of patients has been challenging. Development of processes that enable expansion of human retinal progenitors and the adaptation of these processes to good manufacturing practices is a focus for several laboratories, and there are encouraging signs that this problem will be solved in the near future.

ES- and iPS-Derived Retinal Cells

Utilization of ES or iPS cells as retinal progenitors depends on being able to produce appropriately differentiated and committed progenitors from pluripotent cells. Differentiation protocols to produce neurons are now well established, and protocols that produce retinal progenitors⁶⁹ and photoreceptor-like cells^70,71 have been defined. These protocols generally involve a multistep process taking the ES or iPS cells through the specification of neurectoderm and/or optic cup followed by terminal differentiation, and often involve growing the cells in suspension (or as neurospheres) followed by adherent culture on specific substrates. In addition to being complex, these protocols are time consuming, often proceeding only somewhat faster than the process in vivo. It can take 3–4 months to generate the final, differentiated retinal cells.⁷²

ES-derived retinal progenitor cells can be transplanted into the retina, integrate, and continue to express photoreceptor markers. ES-derived cells transplanted into the crx mouse not only integrate but also restore some light response as measured by b-wave on ERG.⁷⁰ However, the process suffers from poor efficiency; in one report, only 50 cells of a total of 50,000 transplanted differentiated iPS cells survived after transplant in a wild-type mouse retina.⁷³ This low efficiency may be related to decreases in cell viability due to the cell sorting that was performed and or transplantation into normal retinas, which are much less receptive to transplants than injured retinas.

Muller Cells as Progenitors

In lower animals, the retina is capable of complete regeneration even in adults. This argues that either there are multipotent stem cells in the retina or that certain cells of the mature retina can be reprogramed after injury to become multipotent progenitors. In lower animals, both RPE and Muller glia contribute to retinal regeneration after injury. There is a growing body of evidence that mammalian Muller cells in the retina can regress into multipotent cells and differentiate into both neural and glial elements of the retina, at least in rodents. Muller cell lines can be derived from rodent and human retinas that display neural stem cell characteristics, integrating into the retinas of RCS rats and expressing retinal neuronal markers.^74,75 In vivo, mammalian Muller glia respond to injury by up-regulating many of the same pathways that are activated in regenerating retinas in lower animals, but the process does not effectively produce new retina. However, stimulation of these cells in situ with a combination including Wnt and Notch signaling in appropriate sequence seems to facilitate their capacity to regenerate photoreceptors.⁷⁶ The efficiency with which Muller cells or RPE can produce stem-like cells in vitro is low, but the ability of those cells once generated to expand and to redifferentiate into retinal elements is very promising.

Practical considerations for retinal transplants

The underlying premise of stem cells and regenerative medicine is the ability to specifically replace the cells that are lost in disease—for example, to replace photoreceptors in RP patients with rhodopsin mutations or RGCs in glaucoma patients. What, then, are the metrics to define which cell types are optimal for different diseases? Practically speaking, the strategy to treat the loss of a specific cell type with the replacement of that specific cell type may overlook significant opportunities. Biology and medicine remain descriptive sciences, and, in some cases, we may observe that a cell type degenerates in disease but the defect in the system may be driven by a separate cell type which may not be obviously toxic. Many therapeutic approaches outside cell therapy do not address underlying causes and yet provide significant benefits—if a mesenchymal stem cell (MSC) can secrete factors that delay the degeneration of photoreceptors in RP by 50%, this would be a fantastic benefit for RP patients. The metrics for choosing cell types should be focused on safety and efficacy in practice—Do what works.

Safety is a key consideration. Transplanted cells should be capable of migration and limited proliferation as needed to perform their therapeutic effect but should be sufficiently differentiated that they will not regain pluripotency and the capability to form teratomas. One of the attractions of employing a sheet of fully differentiated RPE cells for implantation is the ability to implant a population of cells that are terminally differentiated and not expected to grow, and thus would be expected to have a low potential for aberrant growth or the formation of teratomas. Since RPE cells have a characteristic pigmented appearance and are a monolayer, it is possible to visually inspect these sheets and have a high confidence that no or very few undifferentiated cells are present in the transplant. Suspensions of RPE, even if prepared from terminally differentiated cells, may be stimulated by the detachment and suspension process to proliferate and change phenotype. Implanting the cell as differentiated, confluent monolayers may minimize this.

Insertion of cells into the retina, whether attached to a membrane or as a subretinal bleb, creates the potential for the stimulation of proliferative vitreoretinopathy (PVR). In animals, PVR can be created by injecting RPE suspensions into the vitreous,^77,78 and it follows that RPE cells escaping from the subretinal bleb, surgical wound or the cannula during insertion and removal might create PVR. This risk may be lessened if the transplanted cells have a low capability to become RPE.

Immune compatibility is another major practical consideration for stem cell replacement therapy. Full-thickness transplants of fetal retina into the adult eye without immunosuppression or tissue matching do not result in rejection, and these grafts survive for many years.³¹ It is noteworthy that, as was observed in the transplantation experiments, unmatched ES and retinal progenitor cells survive and are not rejected in the absence of immunosuppression⁷⁹ in rodents. These data suggest that immunosuppression beyond the routine postsurgical anti-inflammatory treatments may be unnecessary. However, the evidence that transplantation can be successful in the absence of immunosuppression has come from situations in which care has been taken to preserve the blood-retinal barrier, and the survival of transplanted cells in the face of the microglia/macrophage activation seen in some retinal diseases may be substantially less.

Integration of transplanted cells into the retina strongly depends on the state of the host retina. Transplantation into an intact, undamaged mature retina yields very low levels of integration, while transplantation into a degenerating or immature retina gives a much higher degree of integration. Strategies that “activate” retina to make it more receptive to donor cell integration have included laser lesions,⁸⁰ mild excitotoxicity, and protease-impregnated polymers.⁸¹ Cells are often transplanted into a subretinal bleb, and the surgical manipulation of creating the bleb and injecting material between the retina and the RPE may serve to activate the retina sufficiently while the injection of cells into the vitreous over the retina may not have that effect and generally leads to poorer integration. Of course, injecting cells into a subretinal bleb limits their dispersal in the retina and may lead to smaller areas of integration. While the assumption that the recipient human retinas in a clinical setting are activated is reasonable, it is not necessarily the case that all activation in animal models is the same and care should be taken in the selection of the activation paradigm.

Clinical trials of retinal transplantation are also challenging, because subretinal implantation trials cannot be masked, and the primary and key outcome is a subjective one. If the transplanted cells are able to home to the target after simple intravitreal administration, such trials may, in some cases, be masked in the same way in which trials of intravitreal drugs are masked. However, if the cells need to be implanted into a subretinal bleb or are implanted attached to a matrix that needs to be positioned on or under the retina, the procedure itself carries significant risk. In this situation, the patients cannot be masked although the evaluators can be masked. The scar from the surgery may reveal group assignment to the examiner. Even in the case of intravitreal injections, the cells may form a vitreous haze that is observable to the examiner. Masking of evaluators may require the use of a reading center and careful construction of the reading center protocols to restrict readers' access to images that might reveal group assignment. It is hoped that the magnitude of the therapeutic effect will be large enough to overcome doubts driven by imperfect masking.

Glaucoma

Retinal ganglion cells

In glaucoma, the retinal neurons that require replacement are the retinal ganglion cells (RGCs). Similar to many other degenerative diseases of the retina, a substantial portion of the cells are lost before visual function is lost and before patients are diagnosed with glaucoma. Replacement of lost RGCs is a significant challenge. First, the transplanted RGCs would need to extend axons down the optic tract to their appropriate terminal connections in the lateral geniculate nucleus (LGN). Transplanted neural progenitors have been reported to invade the optic nerve and grow substantial distances,⁸² opening the prospect that over longer time periods transplanted RGC axons could reach their targets. Second, for visual function, the synaptic connections would need to appropriately reestablish a functional retina-LGN distribution. In humans, plasticity of the visual connections is limited after the critical period early in life. Functional replacement of RGCs via cell therapy will probably require an effective strategy to stimulate plasticity of the visual connections, and such a strategy would have important implications for the treatment of amblyopia as well. Recent work in rats suggests that thalamocortical plasticity can be reactivated in the adult through dark exposure,⁸³ but the applicability of this to humans remains to be determined.

Trabecular meshwork

A separate approach to glaucoma is the correction of the impaired outflow of aqueous humor, leading to elevations in intraocular pressure (IOP) in primary open-angle glaucoma patients. The most potent current drug treatments for glaucoma are the prostaglandins, which act, in part, by stimulating matrix metalloproteinase expression in the TM. Activation of stem cells and tissue remodeling in the TM might have a similar but more potent and durable effect. Stem cells from TM can be isolated and expanded using conditions similar to other stem cells, and these cells show active phagocytosis.⁸⁴ On transplantation back into the anterior chamber, these cells home to the TM and reduce IOP in animal models, presumably via tissue remodeling.⁸⁵ This progenitor population may not be the only progenitor population in the TM, however, as others have identified a progenitor population in the TM that may be precursors to the corneal endothelial cells, and having a very different phenotype¹⁹; whether there are multiple progenitor populations or a single progenitor which can differentiate into either endothelial or TM cells remains to be determined.

Trophic support

While the approaches described earlier have both the potential for the transplanted cell therapy to replace the original dysfunctional host cells and the ability to support host cell survival and regeneration through trophic effects, some approaches rely solely on the trophic effect. Trophic support by transplanted cell therapy is an attractive approach, as there is a large body of evidence that neurotrophic factors can enhance the survival and function of RGCs in the face of a variety of insults and in a variety of glaucoma models. Transplantation of MSCs⁸⁶ or oligodendrocyte precursors⁸⁷ via intravitreal injection ameliorates RGC loss in some glaucoma models. These effects are viewed as trophic and not as replacement of the RGCs. Human umbilical cord-derived stem cells injected into the eye can preserve RGC cell numbers after optic nerve injury.⁸⁸ In these rats, expression of BDNF and GDNF remains elevated in the retina for approximately 28 days post-transplant. Human MSCs can be treated so as to induce the expression of BDNF and GDNF, and these cells when injected into the eyes of rats can protect the RGCs in an optic nerve transection model.⁸⁹

Source Material

Current work on ocular stem cells has utilized the full spectrum of potential sources—cells that originate from ES, iPS, cord blood, and progenitors derived from embryonic or adult tissue. The sourcing of the cells raises ethical issues in a variety of ways and this topic has been well covered by others. Two approaches used for ocular diseases deserve special note. First, surgical excision of donor tissue from living donors, either autologous or allogeneic, is used for corneal limbal transplants and may be used in the generation of cells from RPESC. These procedures carry risks for the donors, and the risk/benefit equation for patients needs to include consideration of the risk/benefit of the donation procedure. Second, utilization of donor tissue from late embryonic sources (as opposed to supernumerary embryos) is being used to generate neural and retinal progenitors. Tissue that is already differentiated into brain or retina has the advantage that these cells are likely to have a lower potential to form teratomas, and, therefore, are lower risk to the patient. Cells derived from embryonic tissues generally do not express high levels of major histocompatibility antigen on their surfaces and so are less likely to be rejected. In the event that processes are successfully developed which enable the infinite expansion of these progenitor cells, beneficial therapies may be developed using a very small number of donations. However, if it is necessary to establish new progenitor populations periodically or if a large number of progenitor banks are needed for tissue-matching purposes, this would create a need and a commercial incentive for donation. Scientists developing such therapies should strive to develop technology allowing the indefinite expansion of cells and to minimize the number of donors needed before advancing to the treatment of larger numbers of patients.

Source material also has an impact on the potential benefit and risk of the transplanted cells and on the scientific assessment of risk. In the case of a transplant where the cells do not persist and are eliminated by the host immune system or some other mechanism, it should be possible to establish safety by measuring adverse events over the period where the cells are present. However, if the cells that are transplanted are expected to persist for long periods, potentially for the life of the patient, a different approach is needed. For teratoma concerns, the animal data suggest that on transplantation, ES cells recapitulate development over a time frame of 4–6 months and a few teratomas appear later.¹ This suggests that if the concern is contaminating undifferentiated cells in the preparation, a safety evaluation needs to be longer than 4–6 months. If the concern is eventual aberrant growth of the persistent cells, longer evaluations may be necessary, likely in primates. In addition, the genomic stability of cells derived from various sources is known to be different, but the significance of those differences to safety is not clear.⁹¹

The eye holds the advantage that the number of cells needed to be transplanted is typically much lower than for other applications. Teratomas most resemble type 1 germ cell tumors¹ and the founder population necessary to establish such a teratoma is thought to be small, perhaps as few as 10 cells. For many stem cell transplantation applications in humans, this implies that the ability to detect contaminating undifferentiated ES cells at a level of 1 in 10⁷ or 10⁸ cells is a difficult challenge. However, some proposed retinal stem cell therapies involve transplantation of a small number of cells attached to a sheet, for example, in the RPE replacement strategies mentioned earlier. Here, since only about 10⁵–10⁶ cells are transplanted and since they are a monolayer on the sheet, 100% visual inspection is possible, and the required level of sensitivity to detect contaminating cells is orders of magnitude lower.

The diversity of approaches in cell therapy means that any given disease may be approached via several fundamentally different but promising stem cell or regenerative approaches, and it may not be readily apparent which ones will provide benefit to patients. With this promise comes a responsibility to evidence-based medicine and rigorous scientific evaluation. The cell therapy field has provided examples of irresponsible or dangerous interventions² although none yet in ophthalmology. Just as the development of effective therapies to improve and restore vision will provide immense benefits to patients, the introduction of unproven therapy will not only fail to benefit patients but will undermine efforts to develop truly effective therapy. There are currently a number of cell therapies being offered to patients with scant evidence for safety and efficacy, and it is important that the scientific and medical community appropriately provide guidance and advice to patients regarding these therapies.

Summary and Conclusions

Cell therapy for ocular diseases is rapidly advancing. The use of limbal stem cell transplants is already providing large benefits to patients with chemical burns and diseases of the corneal surface. Effective generation of replacement cells for the corneal endothelium remains a large unmet need and active area of research. For retinal diseases, human trials are under way for RPE cell therapy, and retinal progenitors that are capable of restoring photoreceptor function are in advanced preclinical testing. It is hoped that these advancing therapies will not only stop the degenerative process but also recover visual function in patients. However, it is unlikely that there is a “one size fits all” solution to retinal degenerative diseases, and it is likely that different progenitor populations will be needed for these different diseases.

Footnotes

Acknowledgments

The author would like to thank Irv Arons for sharing his table of ongoing cell therapy trials in the eye and Mike Niesman, Sally Temple, and Jeff Stern for a critical reading of this article along with their valuable suggestions.

Author Disclosure Statement

No competing financial interests exist.

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