Stem Cell-Based Treatment Strategies for Degenerative Diseases of the Retina

Abstract

Background:

The main cause of progressive vision impairment in retinal degenerative diseases is the dysfunction of photoreceptors and the underlying retinal pigment epithelial cells. The inadequate regenerative capacity of the neural retina and lack of established therapeutic options demand the development of clinical grade protocols to halt the degenerative process in the eye or to replace the damaged cells by using stem cell-derived products. Recently, stem cell-based regenerative therapies are at the forefront of clinical investigations for retinal dystrophies.

Objective:

This article will review different stem cell-based therapies currently employed for retinal degenerative diseases, recent clinical trials, and major challenges in the translation of these therapies from bench to bedside.

Methodology:

A systematic literature review was carried out to identify potentially relevant articles published in MEDLINE/PubMed, Embase, ClinicalTrials.gov, Drugs@FDA, European Medicines Agency, World Health Organization International Clinical Trials Registry Platform.

Result:

Transplantation of healthy cells to replace damaged cells in the outer retina is a clinically relevant concept because the inner retina that communicates with the visual areas of the brain remains functional even after the photoreceptors are completely lost. Various methods have been established for the differentiation of pluripotent stem cells into different retinal cell types that can be used for therapies. Factors released from transplanted somatic stem cells showed trophic support and photoreceptor rescue during early stages of the disease. Several preclinical and phase I/II clinical studies using terminally differentiated photoreceptor/retinal pigment epithelial cells derived from pluripotent stem cells have shown proof of concept for visual restoration in Age-related macular degeneration (AMD), Stargardt disease, and Retinitis pigmentosa (RP).

Conclusion:

Cell replacement therapy has great potential for vision restoration. The results obtained from the initial clinical trials are encouraging and indicate its therapeutic benefits. The current status of the therapies suggests that there is a long way to go before these results can be applied to routine clinical practice. Input from the ongoing multicentre clinical trials will give a more refined idea for the future design of clinical-grade protocols to transplant GMP level HLA matched cells.

1. Introduction

Retinal degenerative diseases (RD) cause progressive visual deterioration stemming from the continuous loss of the components of neural retina, mainly photoreceptors (PR) and underlying retinal pigment epithelium (RPE). The adult mammalian retina has inadequate regenerative capacity, and thus PR or RPE cell death can lead to irreversible vision loss. Age-related macular degeneration (AMD) is the leading cause of blindness in people above 65 years of age in the US​ [1]​. Retinitis pigmentosa (RP) is the most common ​hereditary retinal degenerative disease that affects 1 in 3000–7000 people [2]. Until recently, treatment for most retinal degenerative diseases consisted of approaches that reduce the adverse effects of the damage or slow down the disease progression. For wet age-related macular degeneration (wet-AMD) and exudative symptoms associated with diabetic retinopathy, frequent anti-vascular endothelial growth factor (VEGF) intravitreal injections are approved for use as treatment by the Food and Drug Administration (FDA) [3]​​. Meanwhile, a new ​gene therapy ​has been approved by the FDA to ​treat​​ Leber congenital amaurosis and another rare genetic retinal disease with biallelic RPE65 mutation. However, there do not yet exist effective treatments for the remaining majority of retinal degenerative diseases such as dry age-related macular degeneration (dry-AMD), Stargardt disease, RP and certain other types of inherited retinal dystrophies. ​Attempts have been made to develop novel therapies to regenerate the atrophic or damaged retinal tissue through prolonged administration of neurotrophic factors [4], complement inhibitors [5], immunomodulators [6], ​anti-inflammatory agents [7], visual cycle modulators [8], and stem cells. Among these, the cell mediated therapies are evolving as the most promising therapeutic strategy, wherein the dysfunctional retinal cells can be replaced with healthy and functional cells derived from stem cells. Cell preservation strategies are another option, wherein the trophic factors produced by the transplanted cells will rescue and preserve the existing photoreceptors from damage. Both strategies are advantageous as ​the cells in the inner retina communicating to the brain remain mostly functional during the progression of the disease. ​Among cell replacement therapies for various organs, retinal stem cell therapy is theoretically the most realistic one due to the uncomplicated neuroanatomy, immune privileged status of the eye and easy access of the inner eye for manipulation and imaging. Results from numerous well-established preclinical studies have proven the potential of stem cells in the clinical treatment of RD [9–11].

The main candidates for stem cell mediated retinal repair are retinal progenitor cells (RPC), stem cell-derived photoreceptors/photoreceptor precursors (PR/ PRP), and RPE cells. Compelling evidence from earlier autologous transplantation studies of RPE cells showed that healthy RPE can support photoreceptor rescue and visual functional improvement in human patients [12–14]. However, difficulty in obtaining autologous sources of healthy RPE sheets as well as the possibility of genetic risks made autologous RPE a less desirable candidate. The search for healthier alternatives to autologous RPE cells extended to RPE from foetal or adult donor eye tissue, but the latter option is associated with several feasibility and ethical issues. From the year 2000 onwards, the focus has moved to embryonic stem cell-derived RPE. The design of various protocols to differentiate multipotent embryonic stem cells (ESCs) into RPE and PRs made them a more appropriate candidate for an unlimited supply of the RPE cells. After the discovery of induced pluripotent stem cells (iPSCs), they are found to be a remarkable cell source for obtaining differentiated retinal cell types. Additionally, advances in retinal stem cell transplantation over the past decade have led to the development of stem cell delivery tools, good manufacturing practice (GMP) grade stem cell differentiation and sorting protocols, better immunosuppressive regimens and clinical accessibility of the visual system for non-invasive real time imaging. All these facilitated the development of refined clinical strategies with promising outcomes. The recent Phase I/II clinical trials using cells derived from pluripotent stem cells for retinal degenerative diseases have come up with encouraging results. In this review we will provide an overview of the neural retina, the various degenerative diseases affecting it, stem cell-derived products used for the treatment and the ongoing clinical trials.

2. The neural retina

The retina, the inner lining of the back of the eye is the photosensitive part of the central nervous system (CNS) consisting of complex layers of neurons interconnected by synapses and reinforced by an outer layer of pigmented epithelial cells. The three important layers of the retina are the photoreceptor layer, the bipolar cell layer, and the ganglion cell layer. These three primary retinal layers are further divided into seven layers [15]. The first layer contains the pigment end of the receptor cells. The second layer, the outer nuclear layer (ONL), is formed by the nuclei of the rod and cone photoreceptors. The third layer, the outer plexiform layer (OPL), consists of the dendrites and axons of horizontal and bipolar cells. The fourth layer, the inner nuclear layer (INL), is formed by the nuclei of horizontal cells, bipolar cells and amacrine cells. The fifth layer, the inner plexiform layer (IPL) consists of synaptic connections between the axons of bipolar cells and dendrites of ganglion cells. The sixth layer, the ganglion cell layer (GCL) contains the cell bodies of ganglion cells. The seventh layer, the optic fibre layer (OFL) contains the axons of ganglion cells as they accumulate to form the optic nerve. Blood supply to the retina is facilitated through the central retinal artery and the choroidal blood vessels. Phototransduction is the process through which photons, the elementary particles of light are converted into electrical signals that pass through the second and third layer of retinal neurons which convey the information to the brain.

RPE is a monolayer of extremely specific cells located posterior to the photoreceptor layer and anterior to Bruch’s membrane (BM) which acts as the lining of the choroid. The RPE acts as a boundary between the retina and choroid and is connected to adjacent RPE cells through tight junctions to maintain the blood-retinal barrier. Although the RPE is not a defined part of the retina, it is an essential supporting tissue. The elongated apical microvilli of RPE surround the outer photoreceptor segments. The shedded outer segments from the distal end of the PR (rods and cones) are engulfed by the apical microvilli of RPE cells and phagocytosed by the cells through a daily circadian rhythm. Close interaction between the apical microvilli of RPE and PRs is also essential for the RPE to carry out its other functions which include the elimination of metabolic waste from the retina, the guarding of the retina from stray light, the regulation of water and ion flow, and the recycling of visual pigments for photoreceptors. The polarized distribution of proteins on the RPE is important for phagocytosis and transport of opsin and retinal, a product of vitamin A, to the outer segment. During light absorption, the retinal isomerizes from the 11-cis-retinal form to the all-trans-retinal form and undergoes conversion to all-trans-retinol. Photoreceptors are unable to convert all-trans-retinol back into 11-cis-retinal, so it is transported to the RPE for reisomerization and reprocessed back to photoreceptors [16]. BM is a thin (2–4 μm), acellular, five-layered extracellular matrix present between the RPE and the choriocapillaris. It provides structural support to the eye and is also involved in the exchange of nutrients and waste products between the choroidal blood supply and the RPE complex. Fig. (1) shows a diagrammatic representation of the cross section of a normal human eye.

Figure 1.

Drawing showing section through human eye and a schematic enlargement of retina and choroid on the right.

3. Degenerative disease of the retina

RD are a group of progressive diseases that can lead to total blindness. The most common RDs include age-related macular degeneration (AMD), diabetic retinopathy, Stargardt disease, and retinitis pigmentosa (RP), among which AMD is one of the most prevalent ocular diseases projected to affect 288 million by 2040 [17]. The dry form of AMD is the most common type of AMD, occurring in 80–90% of people [18]. It starts with the deposition of small amorphous extracellular deposits underneath the macular region between the inner layer of the BM and the basal membrane of RPE. Over time, the deposits increase with size, and the resultant inflammation leads to degeneration of RPE and photoreceptors, which eventually culminate in the deterioration of central vision. ​More advanced stages of the disease demonstrate geographic atrophy (GA) with well-demarcated areas of RPE and photoreceptor loss [19]. A subset of patients with drusen will progress to the neovascular form of AMD where abnormal capillaries form and creep into RPE and subretinal areas [20]. Despite the differences in pathological progression of various RD diseases, RPE and/or PR dysfunction is the most common pathogenesis of RD. RP disease-causing genes can be classified into eight main groups based on their role in ocular homeostasis, including vitamin A metabolism, ribonucleic acid (RNA) intron-splicing factors, the phototransduction cascade, structural or cytoskeletal roles, intracellular trafficking, pH regulation, cilia maintenance, synaptic interaction, and RPE phagocytosis [21]. Stargardt disease is an eye disease that causes vision loss in children and young adults. It is usually an autosomal recessive condition caused by mutations in the ABCA4 gene. The defect in this gene will lead to accumulation of a major component of lipofuscin in RPE cells causing degeneration of rods and cones and progressive vision loss [22].

4. Stem cell treatment strategies

4.1. Somatic stem cell-based therapies

Somatic stem cells are not pluripotent but can generate somatic cells specific to a particular tissue. The somatic stem cells that are mainly used for treating RD are mesenchymal stem cells (MSC) derived from bone marrow [23–25], umbilical cord [26], adipose tissue [27–29], and human neural progenitor cells [30]. These cells typically assume a trophic role in stem cell therapy. Although there is no convincing evidence that these cells will fully differentiate into photoreceptors, their ability to rescue the degenerating PRs is well-evidenced. This property is found to be contributed by the paracrine factors released by the cells. Many of these stem cell types also exhibit varying immunomodulatory actions including suppression of immune responses and inflammation [31–33]. They release various immunomodulatory proteins including insulin-like growth factor-1 (IGF-1), class II major histocompatibility complex (MHC class II) antigens, and Th2-related cytokines [34, 35]. This transient dosing approach depends on the viability and secretory potential of the transplanted cells. Since RP disease is caused due to mutations affecting PRs, cell preservation strategies are effective for slowing down the progression of vision loss. However, the preservation strategies are effective only in the earlier stages of disease when adequate PRs are present. Stem cell- derived RPE can also be used as supportive cells to provide trophic support for surviving PR [36]. Early-phase clinical trials with encapsulated RPE cells producing ciliary neurotrophic factors suggested PR protection in patients with RD [4, 37].

4.2. Therapies based on retinal progenitor cells (RPCs)

RPCs are mitotically active multipotent stem cells which are found in the developing neural retina. They are capable of differentiating into neuronal cells of the retina. RPCs isolated from various gestational or postnatal periods of rat models express several developmental markers and differentiate into various retinal cell types [38, 39]. Preclinical studies have demonstrated that retinal precursor cells at the peak of rod genesis when transplanted can differentiate into rod photoreceptors, integrate into the degenerating retina, and form synaptic connections, thereby improving visual function [40]. The first FDA-approved Phase I/IIa clinical trial (NCT02320812) using human retinal progenitor cells (hRPC) demonstrated the acceptable safety and tolerability of hRPC transplantation [41]. This is followed by a Phase IIb study designed to assess effect of a single injection of hRPCs in visual function (NCT03073733) [42]. Long-term safety and efficacy are primary objectives of another ongoing RPC transplantation study initiated by ReNeuron Limited. Preliminary results from this study extrapolates that the rescuing effect of RPCs are mainly contributed by neurotrophic factors rather than direct cell engraftment [43].

4.3. Pluripotent stem cells (PSCs) used for therapies

Pluripotent stem cells have the capacity to self-renew by division and develop into the three primary germ cell layers of the early embryo. Embryonic stem cells are the best example of pluripotent stem cells, which have applications in retinal tissue engineering. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells which can be generated directly from a somatic cell line that has regained the capacity to differentiate into any type of somatic cell in the body. Dysfunction and loss of RPE and PRs are the hallmark of most RD. Without functional RPE, the majority of the overlying PRs eventually die, leading to severe vision loss. Replacing lost or damaged RPE and PRs with similar but healthy cells derived from pluripotent stem cells is now at the forefront of retinal regeneration strategies. Recent clinical studies showed that RPE replacement strategies can delay disease progression or even restore lost vision [44, 45]. Pluripotent stem cell-derived retinal cell products used for preclinical studies and clinical trials are listed in Table 1.

Table 1.

A list of major research articles published based on preclinical animal studies and clinical trials using human pluripotent stem cell-derived retinal cells.

Human pluripotent stem cell (hPSC) types	hPSC derived cells	Preclinical animal studies	Human clinical trials
iPSC	iPSC-PR/PRP	Mouse (Barnea-Cramer et al., 2016; Lamba et al., 2006; Zhu et al., 2017) Rat (Gagliardi et al., 2018)	Nil
	iPSC-RO	Mouse (Mandai et al., 2017a)	Nil
	iPSC-RPE		(Mandai et al., 2017b; Sugita et al., 2020)
ESC	ESC-PRP	Mouse (Decembrini et al., 2014; Gonzalez-Cordero et al., 2013; Santos-Ferreira et al., 2016)	Nil
	ESC-RO	Monkey (Shirai et al., 2016) Rat (McLelland et al., 2018) Mouse (Assawachananont et al., 2014; Iraha et al., 2018) Cat (Singh et al., 2019)	Nil
	ESC-RPE	Monkey (Chao et al., 2017) Pig (da Cruz et al., 2018; Kashani et al., 2018; Koss et al., 2016) Rat (Diniz et al., 2013; Hu et al., 2012; M’Barek et al., 2017; Thomas et al., 2016) Mouse (da Cruz et al., 2018; Schwartz et al., 2012)	(da Cruz et al., 2018; Kashani et al., 2018; Schwartz et al., 2016, 2012; Song et al., 2015)

Abbreviations: ESC: embryonic stem cell, iPSC: induced pluripotent stem cell, RPE: retinal pigment epithelium, PR/PRP: photoreceptor/photoreceptor precursors, RO: retinal organoids

5. Differentiation methods for obtaining retinal pigment epithelial cells and photoreceptors from pluripotent stem cells

5.1. RPE derived from embryonic stem cells

The first report of differentiation of ESCs into RPE-like cells was in 2002 by Kawasaki and c-workers. They used a spontaneous differentiation method to generate “RPE-like” cells from primate embryonic stem cells [46, 47]. Later Klimanskaya et al., in 2004, isolated and characterized RPE cells by spontaneous differentiation of ESCs [48]. Based on these findings, several groups have managed to develop a range of directed and spontaneous, efficient and rapid differentiation protocols by incorporating the fundamental developmental biology [49–52]. In spontaneous differentiation, cells are overgrown as adherent cultures or as embryoid bodies. Pigmented cells from embryoid bodies or adherent cultures can be further plated and enriched [48, 52, 53]. Direct differentiation utilizes defined factors to target the generation of ESCs into RPE cells, which follow a developmental path analogous to the embryonic development of the RPE passing through the optic neuroectoderm, eye-field stage, lineage-committed RPE progenitors, immature RPE, and mature RPE [50]. Variability in cell maturation in in vitro conditions demand further cell purification and selection of pigmented cells before transplantation [54].

In the majority of the protocols, it will take an average of 16 weeks for the differentiation and maturation of RPE from embryonic stem cells. ESC-RPE is morphologically and functionally comparable to the resident RPE, which can expand as a monolayer of typically pigmented hexagonal-shaped cells that develop microvilli [53]; ESC-RPE can also take part in phagocytosis [55].

5.2. RPE derived from induced pluripotent stem cells (iPSCs)

The major benefit of using iPSCs to treat AMD is the possibility of developing a patient-specific therapy able to eliminate the problems associated with immune rejection. Proof of concept for the therapeutic use of a patient’s own iPSC-derived RPE lies in current clinical treatments for AMD. iPSC therapy might also be helpful in patients with genetic diseases, such as Leber’s congenital optic neuropathy, where transplantation could be combined with gene therapy to correct genetic defects inherent to the patients’ own RPE cells. In 2009, the first homogenous RPE cells were produced from iPSCs after exploring techniques related to those used in the differentiation of RPE cells from ESCs [56]. In the same year, another set of factors were developed by Jamie Thomson’s group using Oct4, Sox2, Nanog, and Lin28 to derive RPE cells from iPSCs [57]. These iPSC derived cells showed similar protein expression and gene expression and performed phagocytosis of shredded outer photoreceptor segments like the resident RPE cells. Further studies showed that the RPE generated from iPSCs under defined conditions exhibit ion transport, membrane potential, polarized VEGF secretion, and gene expression profile like those of native RPE. iPSC-RPE were found to be similar to human fetal RPE by proteomic analysis and were capable of phagocytizing outer photoreceptor segments in vivo [58, 59].

iPSC-RPE allows autologous transplantation of cells, thereby reversing any inconsistencies in immune-related compatibility for the patient. Like ESCs, iPSCs also have low MHC class I molecule expression. They do not express MHC class II molecules and share morphological and functional features similar to resident RPE cells [60]. The gene expression profiles are comparable to ESC-RPE, but iPSCs may preserve epigenetic markers of their original cell of source [57]. This is the reason why retinal lineage cells are a better source for iPSC-RPE than fibroblasts, but this would necessitate patient retinal biopsies or fetal retinal sources. Other disadvantages of iPSCs are similar to that of ESCs, such as reduced efficiency of conversion to RPE, lengthy processing periods, and variability between different cell line origins [57].

5.3. Photoreceptors / photoreceptor precursors and neuronal retina derived from pluripotent stem cells

A number of protocols have been established to produce PRs from stem cells, mostly through the formation of retinal organoids (RO) similar to the optic vesicle stage in vitro. Organoids contain most of the retinal cell types, especially PRs arranged in a three-dimensional organized structure [61, 62]. For organoid formation using pluripotent stem cells, aggregation of pluripotent stem cells into embryoid bodies is achieved first, and then basal lamina components are added to direct retinal neuroepithelium differentiation. In another method, neural induction is introduced in embryoid bodies in suspension, and then eye field formation in adherent conditions following the separation of retinal domains to culture in suspension. RO derived from pluripotent stem cells are cut into sheets prior to transplantation. hESC retinal sheets transplanted into the subretinal space of mouse eyes showed signs of PR maturation including formation of light-responsive inner and outer segments and synaptogenesis [63]. In another study, direct integration of graft PRs with host bipolar cells was detected after the transplantation of neural progenitor retina sheets into the subretinal space of an end-stage retinal degeneration immunodeficient rat model [64]. After transplantation into RD nude rats, hESC-derived retinal sheets survived and matured fully to form structured ONLs with inner and outer segments [65]. However, in some studies, visual functional improvement was not achieved using classical evaluation methods, probably due to the low number of grafted cells [45, 65]. Rosette formation will also affect the functionality of PR sheet transplants. To date, only a few protocols to generate PRs are compatible with clinical application. The heterogeneity of the cell types obtained from RO necessitates effective methods to specifically separate PRs using a panel of cell surface markers. Earlier studies for PR transplantation using cell suspension was not highly successful because the investigations suggested that the encouraging observations were due to the cytoplasmic exchange between transplanted and host cells [66–68]. Lakowski et al. used a five cell-surface biomarker panel (CD73+, CD24+, CD133+, CD47+, CD15-) to isolate young rod PRs from mouse ESC-derived RO [69] using fluorescent activated cell sorting (FACS). Negative sorting from day 200 RO was done using CD29 and SSEA-1 for the enrichment of PRs. Due to the drawbacks of the FACS method, the researchers adapted magnetically activated cell sorting (MACS) to separate CD73 surface markers expressing photoreceptor precursors [70, 71]. The enriched cell fraction differentiated along the cone lineage four weeks after transplantation in mice [70]. Further studies and different cell surface marker combinations are needed to find the best method for PR/PRP isolation and enrichment.

6. Current clinical trials using human pluripotent stem cell (hPSC) derived retinal cells

6.1. Human embryonic stem cell-derived RPE (hESC-RPE) clinical trial

Schwartz et al assessed the safety and tolerability of hESC-RPE (MA09-RPE) in patients with advanced Stargardt’s macular dystrophy (NCT01345006) and dry-AMD (NCT01344993). Before transplantation, hESC-RPE was characterised by karyotyping, pathogen, and phagocytosis assay testing. Differentiation and purity evaluation was done by morphological assessment, quantitative polymerase chain reaction (PCR), and quantitative immunostaining for RPE and hESC markers. 5×10⁴ hESC-RPE cells in 150 μL were released as submacular injection into an area in the pericentral macula with compromised RPE and PR. After surgery, structural evidence confirmed the attachment of cells [72]. After 12 months, the visual acuity of seven AMD patients was checked, and it was found that three eyes increased at least 15 letters, one eye improved 13 letters, and three remained stable. Among seven STGD patients, three eyes increased at least 15 letters, three remained stable, and one decreased more than ten letters [73]. An assessment of multicentral trials using the same cells after 4 years showed that structural and functional results are encouraging, with more than half of the treated patients showing persistent advancements in visual acuity with possible cellular engraftment. Adverse events noticed were those associated with the surgical procedure and systemic immunosuppression. After surgery, the transplants seemed to expand with increased pigmentation in the subretinal space except in the region of GA. There was no evidence of substantial expansion of pigmented tissue into the region of GA; instead, pigmented cells were found expanded away from the area of GA [73, 74]. The clinical trial initiated by Song et al. was conducted in four RD patients of Asian origin (two SMD patients and two dry-AMD patients). A low dose injection of hESC-RPEs (50,000 cells per eye) showed visual acuity improvement in three patients at 12 months follow-up [75].

In another study, Kashani and co-workers used an implant termed CPCB-RPE1 (California Project to Cure Blindness-Retinal Pigment Epithelium 1) for the treatment of severe vision loss and GA in five AMD patients (NCT02590692). The implant consisted of a polarized monolayer of hESC-RPE on an ultrathin parylene membrane designed to simulate BM. The patients with little possibility for visual recovery were included in the initial study. The transplant was shown to be safe and well-tolerated, and the subjects who received the CPCB-RPE1 implant showed improvement in visual performance [44].

The procedures of stem cell-based cell therapy for treatment of wet-AMD are more complicated than those for dry-AMD. Because the choroidal neovascular (CNV) membrane must be removed before cell transplantation in wet-AMD, the procedure is associated with a higher risk of massive haemorrhage and retinal detachment. Liu et al. [76] delivered clinical-grade hESC-RPE cell suspension into the subretinal space of three wet-AMD patients to test the safety and feasibility (Clinicaltrials.gov: NCT01691261). Consistent with reports by Schwartz et al. [73, 74], there was no evidence of immunologic rejection, cystoid macular oedema, or retinal neovascularization. Overtime, the hESC-RPE cells tended to form a sheet in the subretinal space. Visual acuity in the treated eyes had improved by 16 letters at 12 months post-transplantation without any adverse complications [9]. In another safety and feasibility study, a hESC-RPE patch on a human-vitronectin-coated polyester membrane was used by Da cruz et al. to implant in two subjects with acute wet-AMD. At 12 months, both patients showed improvement in visual acuity. In both patients, integration of the RPE transplant with host retina and focal improvement in photoreceptor anatomy was seen [77].

Two other Phase I/II clinical studies to treat dry-AMD are in progress: one in China (NCT02755428), and another in Israel (NCT02286089). These studies are estimated to be completed in December 2020 and December 2024 respectively. Details of various ESC-based clinical trials are enlisted in Table 1 and Table 2. In conclusion, ESC-RPEs are a promising candidate to treat RD by providing sufficient ready-to-use healthy RPEs.

Table 2:

A summary of clinical trials for retinal degenerative disease using pluripotent stem cell derived RPEs.

Disease condition	ClinicalTrials.gov identifier & Study Start Date	Pluripotent stem cell type used	Purpose/Objective	Study phase	Study location	Sponsors and collaborators
AMD	NCT01674829 September 2012	MA09-hRPE	To evaluate the safety and tolerability of MA09-hRPE cellular therapy in patients with advanced dry-AMD.	I/II	CHA Bundang Medical Center, Korea	CHABiotech CO., Ltd
AMD	NCT02463344 February 25, 2013	MA09-hRPE	To evaluate the long-term safety and tolerability of MA09-hRPE cellular therapy in patients with advanced AMD from 1 to 5 years following the surgical procedure to implant the MA09-hRPE cells.	I/II	Bascom Palmer Eye Institute, USA Jules Stein Eye Institute, USA UCLA School of Medicine, USA Mass Eye and Ear, USA Wills Eye Institute-Mid, USA Atlantic Retin, USA	Astellas Institute for Regenerative Medicine
Stargardt’s Macular Dystrophy	NCT02941991 January 16, 2013	hESC-RPE	To evaluate the long-term safety and tolerability of hESC-RPE cellular therapy in patients with advanced SMD from 1 to 5 years following the surgical procedure to implant the hESC-RPE cells.	I/II	Moorfields Eye Hospital NHS Foundation Trust, England Newcastle on Tyne NHS Foundation Trust, England	Astellas Institute for Regenerative Medicine
AMD	NCT02286089 April 2015	hESC-RPE	Evaluation of the safety and tolerability of OpRegen-hESC derived RPE cells.	I/II	Retina Vitreous Associates Medical Group, USA Byers Eye Institute, Stanford School of Medicine, USA Retinal Consultants Medical Group, USA West Coast Retina Medical Group, Inc, USA	Lineage Cell Therapeutics, Inc. Cell Cure Neurosciences Ltd.
AMD Stargardt’s Macular Dystrophy	NCT02749734 May 2015	hESC-RPE	To determine the safety and therapeutic effect of sub-retinal transplantation of hESC derived RPE in patients with macular degeneration diseases.	I/II	Southwest Hospital, China	Southwest Hospital, China
AMD	NCT02749734 May 2015	hESC-RPE	To determine the safety and therapeutic effect of sub-retinal transplantation of hESC derived RPE in patients with macular degeneration diseases.	I/II	Southwest Hospital, China	Regenerative Patch Technologies, LLC
AMD	NCT03305029 May 2016	SCNT-hES-RPE Cells	To evaluate the safety and tolerability of SCNT-hES-RPE cellular therapy in patients with advanced dry-AMD.	Interventional	CHA Bundang Medical Center, Korea	CHA University
AMD	NCT03046407 September 6, 2017	hESC-RPE	To assess the safety and efficacy of hESC-RPE transplants to treat dry-AMD.	I/II	The first affiliated hospital of Zhengzhou university, China	Chinese Academy of Sciences
AMD	NCT02755428 January 2018	hESC-RPE transplant-ation	To assess the safety and efficacy of hESC-RPE transplants to treat dry-AMD.	I/II	Beijing Tongren Hospital, Capital Medical University, China	Chinese Academy of Sciences Beijing Tongren Hospital
AMD (wet)	UMIN000011929 August 2013	iPSC-RPE	To assess the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from iPSCs in a patient with neovascular age-related macular degeneration.		RIKEN Center for Developmental Biology, Japan	RIKEN Center for Developmental Biology
AMD	NCT02464956 July 2015	iPSC-RPE	Production of iPSC derived RPE cells for transplantation in AMD.	Observational	Moorfields Eye Hospital, England	Moorfields Eye Hospital NHS Foundation Trust Medical Research Council
AMD	NCT04339764 September 23, 2020	iPSC derived RPE/PLGA transplant-ation	To evaluate the safety and feasibility of subretinal transplantation of iPSC-derived RPE, grown as a monolayer on a biodegradable PLGA scaffold, as a potential autologous cell-based therapy for GA associated with AMD.	I/IIa	National Institutes of Health Clinical Center, USA	National Eye Institute (NEI)

Abbreviations: AMD: age related macular degeneration, SCNT-hES-RPE: human somatic cell nuclear transfer embryonic stem cell derived retinal pigmented epithelial cells, hESC-RPE: human embryonic stem cell derived retinal pigmented epithelium, PLGA: poly lactic-co-glycolic acid, iPSC: induced pluripotent stem cells, GA: geographic atrophy, SMD: Stargardt’s macular dystrophy, RPE: retinal pigment epithelium.

6.1. iPSC-RPE Clinical trial

Following the first hESC-derived RPE clinical trial, the first human clinical trial for autologous iPSC-derived RPE sheet was performed in 2013 in a patient with neovascular age-related macular degeneration [78]. The feasibility of transplanting sheets of RPE cells differentiated from iPSCs and their immune competency was assessed. The major focus of the study was to evaluate the safety and possible adverse events. iPSCs were produced from skin fibroblasts using non-integrating episomal vectors carrying genes: GLIS1, L‐MYC, SOX2, KLF4, OCT3/4. The first patient who was enrolled in the study had a declining visual function despite repeated anti-VEGF injections. In this patient, the neovascular membrane was removed and an autologous iPSC-derived RPE cell sheet was transplanted. Initially, the transplanted sheet was found curled, but it flattened over time. After transplantation, the choroidal vessels became visible, and there was no sign of graft rejection or any evidence of tumour formation. At one year after surgery, the best corrected visual acuity did not improve, but it remained stable throughout. No serious adverse events were noticed until 25 months post-surgery. In the second patient, three aberrations in deoxyribonucleic acid (DNA) copy number (deletions) were observed, and the decision was made to end the study because of the possible effects that can be caused due to the deletions [79]. In another study, Sugita et al. transplanted allogeneic iPSC-derived RPE cells in human leukocyte antigen (HLA) matched patients with exudative age-related macular degeneration. The study used only local steroids, but no immunosuppressants. No abnormal growth in the graft area was observed during the 1-year observation period. The graft was found to be viable with a low immune reaction [80]. Moorfields Eye Hospital (London, UK) is currently enrolling dry-AMD patients (NCT02464956) for an FDA-approved clinical trial. In the United States, the National Eye Institute (NEI) has launched a clinical trial (NCT04339764) to evaluate the safety and feasibility of subretinal transplantation of iPSC-derived RPE as a potential autologous cell-based therapy for GA associated with AMD. In this study, the RPE cells were grown as a monolayer on a biodegradable PLGA scaffold. Details of the iPSC-based clinical trials are shown in Tables 1 and 2. Based on these studies, it can be suggested that HLA-matched allogeneic iPSCs-RPEs would be a safer option to treat RD and is more likely to succeed practically.

7. Retinal tissue engineering

Cell suspensions allow easy delivery using small diameter cannulas that cause minimal retinal injury. Only a small bleb will be formed, and it will be quickly absorbed. Cell suspensions are expected to adhere and form a monolayer over the damaged BM. Although visual improvement and preservation of retinal thickness were shown after injecting cell suspension, cell clumping and failure to form an intact monolayer are some of the main issues associated with this approach. Cell suspension injections lack polarized alignment of RPE which makes them inferior to cells transplanted as a monolayer. Furthermore, cells injected as suspension may be lost due to reflux to the vitreal space and may diffuse through the viscous vitreous humor. Sheer stress created by the cannula can also affect the healthiness of the cells. To overcome these issues, cells grown on biocompatible membranes are being used in many transplantation studies. Cell patches designed in this way are found to be more effective than cell suspensions. Several natural and synthetic polymers are used as scaffolds for growing hPSCs. Such scaffolds may also replace the damaged BM. Cells in a carefully designed scaffold can preserve polarized morphology and provide trophic support. Cell sheets are more advantageous when the field of atrophy is comparatively large. Most of the patches are compatible with specially designed delivery devices which make the implantation more effective. Readily available off-the-shelf patches would be advantageous in cases of severe wet-AMD with sudden vision loss. Ultrathin parylene or porous polyester are the non-biodegradable, synthetic scaffolds currently tested for the preparation of hESC-RPE patches in the ongoing clinical trials.

Poly-lactic-co-glycolic acid (PLGA) is a biodegradable synthetic polymer that was tested in another clinical trial for the preparation of iPSC-RPE patches (NCT04339764). Important natural polymers found to be suitable candidates for retinal tissue engineering are collagen types I, III, and IV; fibronectin; gelatine; matrigel; vitronectin; and laminin [81–84]. A number of biodegradable synthetic polymers, including (PLLA), poly (l-lactic acid), PLGA, PLLA–PLGA copolymer systems, polydimethylsiloxane (PDMS), and polycaprolactone (PCL), have applications as scaffolds in retinal tissue engineering [85–90].

8. Cell-free therapies based on stem cells

Stem cell-derived trophic factors and exosomes are two different emerging types of minimally invasive cell-free approaches to treat retinal degenerative disease. Exosomes are microvesicles with a double phospholipid membrane layer and plays an important role in intercellular communication, transporting their cargo of proteins and RNA from one cell to another through exo- and endocytosis mechanisms [91]. Exosomes are produced by almost all types of cells. They are easy to isolate, manage, and store, and additionally are devoid of the risk of immunological rejection, proliferation, and malignant transformation. Yu et al. [92] have shown that exosomes isolated from MSCs, when administered intravitreally into the degenerative eye, reduced the inflammatory reaction, limited the progression of the damage, and improved visual function. Trophic factors are endogenously secreted protein factors that have autocrine and/or paracrine roles controlling cellular processes such as proliferation, differentiation, and regeneration to maintain overall cell homeostasis. There are several studies in which trophic factors, either singly or in combination, were used in an attempt to prevent the loss of retinal photoreceptors [84, 93, 94]. Further studies are needed to observe the long-term benefits of therapies based on growth factors and exosomes, and to elucidate the mechanism by which they act on retinal cells.

9. Gap areas in the current research and future directions

The research so far has brought a thorough understanding of the mechanism of pluripotent stem cell differentiation into RPE cells and PRs. The preclinical studies have shown enough proof for the recovery of visual function after RPC transplantation and stem cell-derived RPE transplantation. The preliminary data from the ongoing clinical trials have proved the safety and efficacy of cell replacement therapies. Nevertheless, more studies are needed regarding the choice of cells and the nature of biomaterial used for the preparation of the tissue to be implanted. There are multiple ongoing clinical trials using RPE, but PR transplantation is still in its infancy. A co-cultured RPE-PR graft would be ideal for bringing more profound visual improvement, especially for the treatment of advanced RD diseases. Organoid-based approaches and sorting PRs from RO are ongoing studies with great potential [69, 71]. 3D bioprinting of the retina [95] and CRISPR CAS technology to edit defective genes are the higher-level treatment choices underway [96]. Obtaining retinal tissue from lower passage cGMP-grade pluripotent stem cells with normal karyotype and gene expression without any genetic inconsistencies should be of top priority. Allogeneic cells are used in the majority of the ongoing studies and clinical trials. The immunosuppression regime should be standardized to avoid related systemic adverse events. Immunosuppression can be overcome by creating hPSC banks with known human leukocyte antigen (HLA) genotypes. Autologous in vivo-RPE derived from patients using gene-editing tools is another choice to avoid immune rejection. For this, genetic screening to avoid any undesirable changes is necessary. Overcoming the possibility of immune rejection and immunological issues related to the by-products of biodegradable scaffolds are other major issues. Choosing the appropriate scaffold material in compliance with the retinal environment is very important. While using RPCs, the chance of residual undifferentiated cells in the graft or cell suspension is another threat. To avoid this issue, terminally differentiated cells are more desirable. Lower passage hPSC-RPE should be used for transplantation as there are chances of genetic alterations during prolonged passaging. During transplantation, it is tricky to maintain the RPE cell sheet intact without folding. Thus, improved surgical skills based on the architecture of the eye are needed to reduce adverse events. Suitable surgical device is necessary to appropriately place the cells that are grown as an intact sheet. Retinal detachment is a potential issue after transplantation surgeries, but proper surgical device and technique could definitely help in retina recovery and minimize mechanical injury [97]. The drawback of RO is the lack of RPE-PR interaction and the lack of vasculature. In some protocols, the generation of RPE cells is reported, but these cells lack monolayer morphology facing PRs. Another major limitation of RO technology is the heterogeneity of RO formed through different procedures and an inappropriate proportion of retinal cell types produced.

Conclusion

Retinal degenerative disease is a devastating condition causing deterioration of the retina due to the progressive death of its cells. The potential of stem cell therapy to preserve or restore vision in RD diseases is finally falling into place. Preliminary data from the well-designed, registered clinical trials using stem cell-derived products showed promising therapeutic effects. However, there are numerous challenges that remain to be addressed. The safety, quality, and expected efficacy of the cell product to be delivered to the patients should be accurately assessed. Optimization of surgical conditions and immunosuppressive regimes are other milestones. Identifying the right stage of disease for transplantation is an important priority for cell replacement therapies. Making a definite conclusion based on the preliminary data obtained from the current clinical trials is difficult due to the small sample size, heterogeneity in approaches, lack of proper control, and short duration of the study. In the near future, the outcomes of the ongoing multicentric clinical trials will provide statistically significant conclusions to develop improved clinical grade protocols for replacement therapies in the eye.