hESC Derived Subretinal Implants

Figure 1: Eye Anatomy: Focus – Fovea, Retina
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Image Source: [9]

Age Related Macular Degeneration, (AMD), is a visual disorder which disrupts function in the macula of the retina. The center of visual field is located in the macula; it is critical for vision sensitive tasks such as reading, and driving. There are two known forms of AMD; both of them affect the Retinal Pigmented Epithelium cells, (RPE) [2]. Dry AMD leads to severe atrophy of RPE cells, eventually causing malnourishment of photoreceptors. Wet AMD occurs when a growth of blood vessels from the choroid expands to the center of the visual field. The macula is normally isolated from blood vessels in order to allow for maximum light to penetrate the retina. Macular degeneration patients demonstrate increased performance in visual tasks when subjects undertake a subretinal implantation of RPE cells. The procedure involves deriving RPE cells from Human Embryonic Stem Cells, (hESC). This approach is remarkable since it allows for the selection of young, healthy RPEs which would otherwise be difficult to obtain from a donor. The present delivery system for implantation in clinical trials involves injection of RPE cells with the use of rigid materials including a stiff cannula. Due to the physical properties of RPE cells, this method leads to clustering and damage of the implanted tissue, as well as risks of retinal injury [1]. Novel technologies for subretinal implants are being trialed in order to reduce hazards involved with the treatment.

1.1 Age Related Macular Degeneration

1.1a Development

The development of AMD is characterized by loss of RPE cells in the macula preventing photoreceptor nourishment. The condition is more prominent in individuals age 50 and older. There are three different stages in the disorder: early, intermediate, and advanced. The stages are classified using drusen as a form of measurement. Drusen is an accumulation of yellow extracellular debris which protrudes from RPE cells [7]. It can be visualized using a dilated eye exam; the amounts of drusen increase as the disease progresses. The early stage is illustrated by small and medium drusen debris, the intermediate stage by medium and large drusen debris, whereas the advanced stage is characterized by deterioration in vision. Drusen is critical because most patients with AMD do not show symptoms until the advanced stage of the condition [9]. Therefore, it is essential that patients benefit from treatment, therapy and counselling as early as possible.

1.1b Dry AMD

Dry AMD is the most common form of the condition, affecting 90% of patients. Most patients don’t show any symptoms until the advanced stage. Therefore, eye care professionals use the dilated eye exam to check for drusen. In the advanced stage, patients will notice geographic atrophy, (Figure 2), a symptom characterized by blurred spots in the center of the visual field, (macula) [9]. As the disorder develops, these spots become greater in size, preventing individuals from performing daily tasks such as reading, driving, and cooking.

Figure 2: Geographic Atrophy
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Image Source: Trial X

1.1c Wet AMD

It is important to keep in mind that all wet AMD cases develop from dry AMD. The condition is only prominent to 10% of patients with AMD. Fragile blood vessels protruding from the choroid begin to grow beneath the macula. These blood vessels are easily injured leading to the release of blood; swelling occurs in the macula afterwards. Unlike dry AMD, symptoms appear rapidly and damage can lead to scarring of the retina. Wet AMD can be treated by a variety of methods including the use vascular endothelial growth factor, (VEGF), inhibitors. Photodynamic therapy and laser surgery are also valid methods used to treat wet AMD [9]. Nevertheless, the use of laser surgery can lead to side effects such as loss of healthy tissue surrounding the emerging blood vessels as well as an increase in blurred vision.

1.1d Physiological Significance of Drusen

Drusen is a significant indicator of early AMD due to the fact that it determines the health of RPE cells. RPE cells are normally responsible for accumulating extracellular debris. The exact method by which drusen develops is not well understood. However, it is evident that this type of debris increases greatly in patients with AMD, [7] [9]. This suggests that drusen is formed by the lack of healthy RPE to remove extracellular debris. Therefore, research involving restoration of RPE cells is crucial to benefit people with macular degeneration.

1.2 Cellular Mechanisms

1.2a Human Embryonic Stem Cells

Embryonic stem cells are derived from embryos, eggs fertilized in vitro, and cultured in a cell culture medium [3]. Although the production of hESC lines is highly inefficient, successful specimens are able to multiply for periods greater than a year. One of the critical challenges to maintain hESC is to keep the cell lines from differentiating. Strategies such as sub-culturing, (the segregation of cell lines as they develop), prevent the hESC lines from differentiating as long as they remain in the proper culture medium. Determining the expression of transcription factors, (ex. OCT 4 and Nanog), [2] [3], as well as cell surface markers specific to hESC are crucial ways to identify if a cell line remains undifferentiated. The relevance of hESC research is outstanding, allowing for the understanding of human development as well as application in medical therapies.

1.2b Medical Applications of hESC

Pluripotency is one of the most fascinating properties of hESC allowing for their differentiation into specific cell tissue. This quality of hESC allows for the development of therapeutic procedures involving the implantation of young and healthy cells into injured tissues. The first clinical trials of hESC were approved by the Food and Drug Administration in 2009, [4]. The embryos were derived from fertility clinics with the consent of donors. This was a milestone in hESC research due to past conflicts with respect to ethical concerns. Patients with spinal cord injury and complete paralysis below vertebrae 3-10 were recruited to take part in the study. Surgeons injected oligodendrocytes derived from hESC into the spine of several patients. Oligodendrocytes are speculated to take months to years before any sign of recovery is detectable. The approach discussed above is being used to differentiate hESC into various cell types.

Figure 3: Development of RPE Cells from Induced Pluripotent Stem Cells (iPSCs)
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Image Source: [15]

1.2c Retinal Pigmented Epithelium Cells

Retinal pigmented epithelium cells, (RPE), are important for the maintenance of photoreceptors through various biochemical interactions. A few of these processes include the recycling of photopigments, metabolising vitamin A, and phagocytosis of the outer segments in photoreceptors. The loss of these RPE impairs photoreceptor activity leads to the development of diseases such as age related macular degeneration. Accumulation of drusen is caused by decreased levels of RPE; symptoms only become apparent during the advanced stages of AMD. Differentiation variables such as growth factors are used to derive RPE from stem cells. Buchholz et al. remove fibroblast growth factor, (bFGF), in Induced Pluripotent Stem Cells (iPSCs) for 35 days to induce RPE differentiation [15]. (See Figure 3).

1.3 Subretinal Surgery

1.3a Overview

More clinical trials have been conducted in various fields of research since 2009 when the FDA permitted the use of hESC. The first paper to describe transplant of hESC-derived cells into human patients was produced by Schwartz et al. in 2012. They derived RPE cells which were then transplanted into individuals suffering from advanced dry AMD, [2]. Improvement in vision was examined by placing patients through visual acuity and visual field tests.

Figure 4: Protecting Cannula Protruding from an Injection Instrument
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Image Source: [19]

1.3b Injection Surgery

The current surgical method of implantation involves injection of hESC into fragments of the sub-macula which are still functional. This step is crucial because it allows for the development of RPE in areas that still contain working photoreceptors. Schwartz et al. take advantage of optical coherence tomography in order to locate living tissue. They use cannula in order to transplant the RPE onto the desired zone, (Figure 4), a task that requires great dexterity. The instrument`s tip is criticized to be quite stiff, preventing surgeons from achieving accurate surgeries, (Figure 5). Issues involving hESC-derived cell transplantation include the potential to induce hyperproliferation, abnormal growth, or rejection of RPE due to immune response in individuals, [2]. None of these factors were observed in patients throughout four months. Schwartz et al. are currently attempting to expand their range of patients to benefit individuals with early and intermediate dry AMD. They theorize that the effects of therapy should increase if patients are treated in the early stages of the condition. These individuals have minimal amounts of drusen as well as the healthiest and greatest amount of photoreceptors. Experts argue that significant developments in surgical procedures are necessary to take full advantage of hESC-derived cell transplants [1] [2] [15].

Figure 5: Instrument`s Tip
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Image Source: [19]

Figure 6: Platform Device Diagram
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Image Source: [1]

1.3c Platform Device Surgery

A study produced by Hu et al. illustrates a novel technology which could alleviate issues associated with current subretinal implant procedures. The authors invented a platform device which is able to deliver a 10µm thick monolayer composed of RPE derived from hESC, (Figure 5). The platform device was trialed on rat models to determine possible hazards. Current clinical trials use injection methods in order to deliver RPE. This type of procedure is argued to be highly inefficient, since RPE are required to polarize as a monolayer in order to retain function. Thus, the injection can lead to the formation of RPE clumps, [1]. Furthermore, this cluster of RPE cells can eventually die or become cause immunological reactions. In mouse models, the injection tools are stiff enough to cause retinal detachment, eventually causing retinal folding, [1]. Currently, surgeons need to aim at regions which still contain healthy photoreceptors. The device allows the surgeon to perform the procedure with more accuracy and comfort by covering the whole subretinal area. Furthermore, histological imaging demonstrates minimal damage to the retina and the RPE monolayer post-surgery (Figure 6). Black arrows indicate damage in the peripheries of the structure.

Figure 7: RPE Platform Device Surgical Histology
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Image Source: [1]
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