The Eye

The Eye, Volume Four, the latest release in the Stem Cell Innovation in Health and Disease series, addresses the recent data accumulated on the potential applications of stem cells to treat eye diseases. This volume highlights the recent development of cutting-edge in vitro and in vivo research tools and approaches, including human and murine organoid cultures, genetic editing in vitro and in vivo, human iPSC models of disease, haploid cells for genetic as well as compound screening paradigms, genetically engineered mice, and stem cell transplantation to cure eye diseases.

The volume is written for researchers and scientists in stem cell therapy, cell biology, regenerative medicine and organ transplantation, and is contributed by world-renowned authors in the field. This is a timely and fascinating collection of information and new discoveries that provides a contemporary snapshot album on the fast-moving field of regenerative medicine and stem cell therapeutics.

• Provides cutting-edge research to understand stem cell functions used in disease treatments of the eye
• Develops processes to bring stem cells from bench to bedside
• Includes up-to-date references on stem cell biology and function in common eye diseases and disorders

• Language: English
• Publisher: Academic Press
• Release date: Mar 31, 2023
• ISBN: 9780323983167

Book preview

Chapter 1

Stem cell-based organoid cultures as innovative approaches for ocular repair and regeneration

Igor O. Nasonkin¹ and Lawrence J. Rizzolo², ³,    ¹Phythera Therapeutics, Inc., San Leandro, CA, United States,    ²Department of Ophthalmology and Visual Science, Yale University, New Haven, CT, United States,    ³Department of Surgery, Yale University, New Haven, CT, United States

Abstract

Regenerative medicine approaches to improve and/or restore vision attracted a lot of attention in the past 20 years. Approaches for cell transplantation were advanced by the discovery of ocular tissue stem cells and protocols to differentiate pluripotent Stem Cells Internationalo different ocular tissues. While these discoveries are exciting and potentially promising for restoring sight in human patients, so far the actual clinical impact turned out to be very limited. For many innovative approaches, the big challenge was translating discoveries made in a dish and/or in the animal (usual rodent) models to patients. Specifically, the promise of that progress has not yet translated to efficacious therapies for patients with impaired vision. This has tempered the initial excitement of differentiating cells in a dish or identifying populations of endogenous stem cells that respond to injury/stimuli with limited proliferation. Here we dissect the current approaches under investigation to treat vision loss with the help of stem cells, and critically discuss their limitations and promise. We focus on the results, which have been tested in the clinic, as well as highlight other innovative technologies, which may or may not find a way in the clinic but have a potential for clinical translation. We emphasize the critical importance of bridging the bench discoveries and clinical translation, which is the bottleneck where many ideas fail. We hope to give readers the basis for critical thinking when discussing the potential of stem cells to repair and/or restore vision. Understanding the real limitations will enable us to focus efforts and funding on realistic technologies, and gradually incorporate more innovative ideas to translate such approaches to the clinic.

Keywords

Ocular transplantation; blood-retinal barrier; cornea; keratoplasty; stem cell-based organoid cultures; regenerative medicine

Regenerative medicine approaches to improve and/or restore vision attracted a lot of attention in the past 20 years. Approaches for cell transplantation were advanced by the discovery of ocular tissue stem cells and protocols to differentiate pluripotent Stem Cells Internationalo different ocular tissues. While these discoveries are exciting and potentially promising for restoring sight in human patients, so far the actual clinical impact turned out to be very limited. For many innovative approaches, the big challenge was translating discoveries made in a dish and/or in the animal (usual rodent) models to patients. Specifically, the promise of that progress has not yet translated to efficacious therapies for patients with impaired vision. This has tempered the initial excitement of differentiating cells in a dish or identifying populations of endogenous stem cells that respond to injury/stimuli with limited proliferation. Here we dissect the current approaches under investigation to treat vision loss with the help of stem cells, and critically discuss their limitations and promise. We focus on the results, which have been tested in the clinic, as well as highlight other innovative technologies, which may or may not find a way in the clinic but have a potential for clinical translation. We emphasize the critical importance of bridging the bench discoveries and clinical translation, which is the bottleneck where many ideas fail. We hope to give readers the basis for critical thinking when discussing the potential of stem cells to repair and/or restore vision. Understanding the real limitations will enable us to focus efforts and funding on realistic technologies, and gradually incorporate more innovative ideas to translate such approaches to the clinic.

Ocular therapies leveraging stem cell technologies are gaining momentum because of the urgency of finding a cure for vision loss, and because of the accessibility of ocular tissues, enabling cell transplantation and ocular injections. The devastating retinal degenerative diseases such as Age-related macular degeneration (AMD), Retinitis Pigmentosa (RP), and Stargardt’s disease (SD) lead to permanent blindness and are costly to manage (Access Economics, prepared for AMD Alliance International: The Global Economic Cost of Visual Impairment 2010; Frick et al., 2010; Ferris and Tielsch, 2004; Rein et al., 2006). The urgency of finding new treatments, based on stem cell technologies, is propelled by the fact that there are currently no satisfactory treatments for these blinding diseases available. The human stem cells are viewed by many as an excellent source of tissue-specific cells (including corneal and retinal cells) for the replacement of ocular cells impacted by degenerative conditions, ocular trauma, and aging.

There are two principally different alternative strategies for repairing vision with a help of stem cells. There are many flavors of either two, but in the big picture, there is a replacement strategy and a regeneration and repair strategy.

The replacement strategy relies on the derivation of cells (impacted by the ocular disease, for example, retinal pigment epithelium (RPE), photoreceptors, corneal epithelium) in a dish, expanding them ex vivo and then surgically transplanting them back into the eye. A variation of this approach (still, under the umbrella of replacement) is a derivation of the whole tissue or a layer of cells (e.g., cornea, or retina) and then surgically transplanting the derived tissue into the eye. Yet another variation is expanding the patient’s ocular cells, or cadaver ocular cells (e.g., RPE or corneal limbus cells), and transplanting them into the patient’s eye with anticipation that such cells will functionally replace the patient’s cells and thus improve vision. The common theme is the need for transplantation and the functional integration of such cells. The frequent issues, which basic researchers sideline when describing a novel and exciting potential treatment are (1) sterility of cells (strictly regulated by FDA for biologic products), (2) scalability of the method (working in a small experiment in rodents but then failing during translation step), (3) surgical approaches, which are frequently different in rodent models versus human ocular surgeries, (4) immunosuppression protocols, which almost always cannot be translated from rodents/rabbits to human patients. There is an array of additional problems, which collectively render a great many potentially promising approaches completely useless in the clinic. The general lack of understanding by basic researchers of how the efficacy and safety and reproducibility are justifiably critical for the FDA for permitting clinical testing (not even converting such therapies to routine clinical protocols for treatment of blind people) generated a lot of froth around stem Cell Replacement strategies, glorifying each and every Cell Replacement method and ignoring the realism about the limitations dictated by clinical translation.

The regeneration and repair (R&R) strategies count on inducing the endogenous limited pool of stem/progenitor cells (present in most tissues including the ocular tissues such as the cornea, neural retina, and RPE) to proliferate and self-repair the tissue (i.e., cornea, retina). The critical difference with the 1st approach is no need for developing a transplantation device, thus solving one of the major critical bottlenecks in translating a promising finding to the clinic. As discussed, most cell/tissue replacement methods require a robust surgical tool, and such therapies are usually pitched to FDA as a combination (combo) method (device + biologic) thus making the regulatory phase even more challenging and therapy less feasible. The R&R approaches circumvent this critical bottleneck but impose a different and very critical limitation, which is the need for patient’s tissue, and in fact, ocular niches (e.g., choroid-Bruch’s membrane-RPE, RPE-photoreceptors, cornea) to be present to be able to stimulate these niches to undergo a self-repair process. Once the niche (e.g., RPE-choroid) is in a terminal stage of degeneration, it is obvious that any potential pool of stem/progenitors present there and capable of repair is gone, together with any chance of a positive therapeutic outcome. Collectively, this limits any plausible applications of R&R technologies to ocular diseases in the initial stage of pathology onset, when niches (hence, stem/progenitor cells) are still present. On the contrary, the replacement strategies are frequently pitched as technologies, which can be used even at a late stage of ocular disease (e.g., when extensive corneal damage or complete degeneration of RPE or photoreceptors is the case). Another critical difference is no need for storing and characterizing the cells (where tumorigenicity, cell identity, sterility, viability, and toxicity are some of the many issues derailing the overall efficacy of these therapies). We will discuss the specific limitations of these approaches below, and the above paragraphs only highlight the major differences between the replacement and R&R" approaches even without diving into their clinical feasibility.

The anatomy of the human eye (Fig. 1.1A) presents a very good opportunity for therapies requiring ocular transplantation or/and injection of cells. An array of well-established high-resolution noninvasive monitoring techniques of both the anterior and posterior segments is available to validate the injection and follow the progress of cell grafts. The eye is an anatomically closed organ due to the presence of the blood-retinal barrier (BRB) and ocular pressure. Both the BRB and ocular pressure enable the intra-ocular injection of cells, drugs, and/or biomaterials and facilitate their maintenance in the ocular space, thus reducing the chances of systemic biodistribution throughout the body and improving efficacy and safety.

Figure 1.1 Structure of the retina. (A) The retina is a two-layered cup formed by the retinal pigment epithelium (RPE) monolayer (black) and the neurosensory retina (red). The subretinal space is a potential space that lies between them. The choroid lies between the sclera (orange) and the RPE. The ciliary body and iris are shown in blue. The needles show two surgical approaches to the subretinal space. The trans-scleral approach is used in rodents due to the large lens. To insert the implant, the subretinal space is enlarged by injecting fluid to create a localized retinal detachment. RPE removes the subretinal fluid to reattach the retina. The pars plana approach to the macula is used in humans. The box is enlarged in panels (B and C). (B) A cartoon of the five major classes of neurons is superimposed on a toluidine-blue stained section of epoxy-embedded retina.

Light goes first through the cornea (a semitransparent tissue with several highly organized layers of cells), and after passing through the transparent lens and the jelly-like vitreous body, penetrates the retina, where it crosses several layers of the neural retina to reach photosensitive cells (photoreceptors) and the photons are converted into electric impulses. These electric signals are then passed synaptically and backward through the retinal layers from photoreceptors to retinal ganglion cells, which then send these signals via the RGC projections (forming the optic nerve) to the brain centers processing this visual information (LGN, SC).

Here we separated the ocular diseases into several types based on the type of critical ocular tissue affected (cornea, retina/RPE, and the optic nerve) and review the therapeutic opportunities involved in the stem cells in this order. We do not discuss the lens (which can now be made out of plastic and replaced using the routine surgical procedure) and the cell-free vitreous body.

1.1 Cornea

The cornea consists of several (5–7) layers of epithelial cells about 50-µ thickness (Meek and Knupp 2015), and the stroma (keratinocytes), which accounts for 85%–90% of corneal thickness (Meek and Knupp 2015). The Bowman’s layer separates the corneal epithelium from the stroma; the Descemet’s membrane is located below the layer of the stromal cells and is composed of endothelial cells. The transparency of all layers of the cornea is critical for preventing light scattering and enabling unperturbed light transmission resulting in reaching the posterior segment (the neural cells at the back of the eye).

The cornea is avascular but is heavily innervated The loss of sensory innervation impacts vision in a condition known as neurotrophic keratopathy, leading to corneal ulcers, perforation of the stroma, and defects in corneal epithelium, collectively, heavily impacting vision clarity (Yang et al., 2018). Thus, the need for perfect optical transparency and innervation makes any cell transplantation strategy a significant challenge. This is especially true for nonautologous cells, which have much higher chances of being rejected unless the right immunosuppression regimen is used. Any case of rejection creates a tissue scar, which induces fibrosis impacting both the corneal transparency and innervation (Eslani et al., 2017).

One critical difference between the task of repairing or replacing the cornea versus repairing the retina/RPE or the optic nerve is obvious: the whole cornea can be replaced by a routine surgical procedure (keratoplasty) (Tan et al., 2012), while the whole retina or the optic nerve cannot be replaced. This difference puts substantially less pressure on the need for developing corneal cell transplantation or/and corneal regeneration approaches involving stem cells. Nevertheless, here we describe some remarkable examples of how corneal stem cells can be practically used in clinics to drastically repair corneas, which sustained some major damage. This procedure has been developed because of the presence of quiescent corneal limbal stem cells on the periphery of the cornea (Ksander et al., 2014). This technique simplifies corneal repair and circumvents the need for immunosuppression. The two variants of this technique leverage the ability of quiescent corneal limbal stem cells to re-enter the division and repair the damaged corneas when the limbal cells are forced to divide. One procedure is called simple limbal epithelial transplantation, or SLET (Sangwan et al., 2012; Shanbhag et al., 2019; Basu et al., 2016), and is based on taking a biopsy of a patient’s limbal tissue from the eye, not affected by corneal limbal damage, dissecting into tiny pieces, and applying directly onto damaged corneas, with striking positive results. This procedure skips culturing the limbal stem cells (which has the potential to introduce changes in cells leading to a decrease in their proliferative potential and more) and shortens the time for therapy by expanding the cells directly on the patient’s corneas. There is no need for immunosuppression in the SLET procedure because of the use of the patient’s cells. The SLET procedure has been robustly tested in clinics with some remarkable positive results. A variation of SLET is the expansion of the corneal limbal biopsy-derived stromal cells in a dish before grafting them into the area of corneal damage (Basu et al., 2014). The technique leverages the knowledge about the potential of cultured stromal stem cells to differentiate and repair damaged corneas (Du et al., 2005, 2009).

Collectively, there is a clinically tested and working, truly stem Cell Replacement-based approach to repairing severely damaged corneas. This procedure complements the routine surgical (traditional) approach of replacing corneas, using the cadaver (donor) corneas and immunosuppression.

The demonstration of real clinical efficacy and feasibility of the autologous limbal stem cell-based therapy of repairing corneas facilitated the development of approaches leveraging the human cultured embryonic (pluripotent) stem cell (hPSCs)-derived corneal tissue (Kosheleva et al., 2016; Mathan et al., 2016; Higa et al., 2014, 2019, 2020a; Foster et al., 2017; Singh, Tiwari, et al., 2021; Susaimanickam et al., 2017; Theerakittayakorn et al., 2020; Ida et al., 2022). These corneal stem cell culture technologies carry a promise of avoiding performing a biopsy from a patient’s undamaged cornea or remaining part of the cornea to repair the corneal surface. Moreover, such technologies enable the establishment of the cGMP (which stands for current good manufacturing practice) banks of human corneal stem cells validated and tested for identity, purity, and mycoplasma/bacterial/fungal contamination. This, in turn, facilitates the standardization of cell therapy procedures, as required by the US Food and Drug Administration. This approach is especially critical when both corneas of a patient are badly damaged and isolation of autologous corneal limbal stem cells is thus not feasible. However, it is critical to remain realistic on the feasibility of maintaining the efficacy (therapeutic potential) of corneal stem cells during the expansion of the cultures. There are no methods of precisely maintaining the epigenome (in addition to genetic integrity) of the cells during large-scale expansion. Because stem and progenitor cells inevitably change during large-scale expansion and passaging (Yang et al., 2018; Liu et al., 2020; Oliveira et al., 2014; Alves et al., 2010; Keller and Spits 2021), their epigenetics is impacted by every passage (stem cell senescence). This, in turn, diminishes their therapeutic potency (the ability to acquire the needed identity after grafting and seamlessly integrating into the damaged cornea). Focusing on the large-scale expansion of stem cells almost inevitably leads to the accumulation of chromosomal and epigenetic abnormalities, selecting for rapid growth and increasing chances of tumorigenicity after transplantation. Limiting the number of passages (after establishing pure corneal stem cell organoid/spheroid culture) before banking the cells is a key to solving this real problem. The second important factor limiting the potential of this technology is the nonautologous nature of these cells, which dictates the need for (at least limited) immunosuppression. However, the cornea is a partially immune-privileged tissue and thus corneal grafts may not need systemic immunosuppression (which increases tumorigenicity) (Akkaya et al., 2019). The tremendous advantage of corneal cell therapies is the ability to administer the needed immunosuppression via eye drops. Therefore, the technology of corneal transplantation has been worked out and thus the nonautologous limitation of banked corneal stem cells could be easily solved. The feasibility of the allogeneic SLET (when a corneal limbal tissue biopsy from a cadaver donor eye is used as a cell source) (Riedl et al., 2021) opens the opportunities for generating the banks of nonautologous corneal stem cells for clinical applications. At the same time, just like with the availability of corneal transplantation technology, the allo-SLET presents a challenge to the whole idea of banking the corneal stem cells The allo-SLET provides the authentic limbal stem cells to the grafts without introducing a passaging-expansion step (which, as we discussed, does impact the epigenetics and therefore, the therapeutic potency and efficacy of cell preparation). One important argument for corneal stem cell banking is the reduction of chances of potential contamination in transplanted cells. However, the danger of introducing contamination during the all-SLET procedure is no different than during corneal transplantation (which has been worked out). Collectively, due to the availability of the abovementioned techniques, corneal organoid/spheroid culture needs further proof of being a reliable alternative when the risks are considered (e.g., rejection, potency/identity, and of course the considerations of the cost of cGMP-grade cell expansion, banking, validation, and characterization, which are almost always ignored in academic publications).

Last, it is important to define what exactly is being cultured in 3D corneal aggregates, whether these cells are: (1) spheroids (the multicell aggregates of dissociated tissue, enabling maintenance of stemness and promoting cell division and expansion, for example, corneal limbal stem cells (Singh et al., 2021)), or (2) organoids (which differentiate from pluripotent stem cells following the same developmental pathway as tissue during embryogenesis) and represent, at least partially, the 3D composition of cell layers within the differentiating/differentiated organ, for example, the cornea (Foster et al., 2017). Corneal and other organoids (e.g., retina) are tissues in a dish and hence, the spheroid cultures enriched in certain types of cells within the organoids could also be established from such tissues in a dish (e.g., limbal stem cell spheroids from hPSC-cornea). These differences have been extensively discussed in the literature (Kim et al., 2023; Meenen et al., 2021; Torras et al., 2018) but are still frequently being used interchangeably (Higa et al., 2020b). Corneal stromal/limbal, stem cell spheroid cultures are designed for expansion of the cells for the abovementioned therapeutic applications, and the cells within such aggregates have more or less uniform characteristics and markers—at least they are highly enriched for stem/progenitor cells carrying limbal stem cell markers (Singh, Tiwari, et al., 2021; Mei et al., 2014; Javid 2021; Mathan et al., 2016; Kim et al., 2017). This relative uniformity facilitates the transition of cell therapy technologies to clinical applications, where IND’s CMC section needs to describe the identity and purity of any cell preparation considered for cell therapy. In contrast, the organoids (tissue in a dish) carrying exclusively corneal tissue or many ocular tissues (including cornea, retina, and even RPE) (Foster et al., 2017; Isla-Magrané et al., 2021) are a cheaper and sometimes better alternative to animal models for studying; (1) ocular development, (2) ocular pathologies (when organoids are derived from patient’s cells, and (3) for drug