Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma.

Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm's canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.

Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control?

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Stem cell transplantation rescued a primary open-angle glaucoma mouse model.

Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if transplanted stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.

Two-step induction of trabecular meshwork cells from induced pluripotent stem cells for glaucoma.

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.

Promising Approach in the Treatment of Glaucoma Using Nanotechnology and Nanomedicine-Based Systems.

Glaucoma is considered a leading cause of blindness with the human eye being one of the body's most delicate organs. Ocular diseases encompass diverse diseases affecting the anterior and posterior ocular sections, respectively. The human eye's peculiar and exclusive anatomy and physiology continue to pose a significant obstacle to researchers and pharmacologists in the provision of efficient drug delivery. Though several traditional invasive and noninvasive eye therapies exist, including implants, eye drops, and injections, there are still significant complications that arise which may either be their low bioavailability or the grave ocular adverse effects experienced thereafter. On the other hand, new nanoscience technology and nanotechnology serve as a novel approach in ocular disease treatment. In order to interact specifically with ocular tissues and overcome ocular challenges, numerous active molecules have been modified to react with nanocarriers. In the general population of glaucoma patients, disease growth and advancement cannot be contained by decreasing intraocular pressure (IOP), hence a spiking in future research for novel drug delivery systems and target therapeutics. This review focuses on nanotechnology and its therapeutic and diagnostic prospects in ophthalmology, specifically glaucoma. Nanotechnology and nanomedicine history, the human eye anatomy, research frontiers in nanomedicine and nanotechnology, its imaging modal quality, diagnostic and surgical approach, and its possible application in glaucoma will all be further explored below. Particular focus will be on the efficiency and safety of this new therapy and its advances.

Human Trabecular Meshwork Progenitors.

Trabecular meshwork (TM) cells are a group of progenitors that have the ability to become adipocytes, chondrocytes and endothelial cells. Therefore, those adult corneal progenitors may be used as an effective therapy for trabecular meshwork diseases such as glaucoma, corneal endothelial dysfunctions such as blindness due to corneal endothelial dysfunction, and similar diseases. In order to promote the understanding of human trabecular meshwork progenitors, this article reviews human trabecular meshwork progenitor therapy and discusses its potential applications for curing human eye blindness.

Trabecular meshwork cells are a valuable resource for cellular therapy of glaucoma.

Trabecular meshwork (TM) contains a subset of adult stem cells or progenitors that can be differentiated into corneal endothelial cells, adipocytes and chondrocytes, but not osteocytes or keratocytes. Accordingly, these progenitors can be utilized as a cell-based therapy to prevent blindness caused by glaucoma, corneal endothelial dysfunction and other diseases in general. In this review, we review in vitro expansion techniques for TM progenitors, discuss their phenotypic properties, and highlight their potential clinical applications in various ophthalmic diseases.

Five-Year, Prospective, Randomized, Multi-Surgeon Trial of Two Trabecular Bypass Stents versus Prostaglandin for Newly Diagnosed Open-Angle Glaucoma.

PURPOSE: To evaluate 5-year safety and efficacy of 2 trabecular micro-bypass stents versus prostaglandin as initial stand-alone treatment for newly diagnosed, treatment-naive primary open-angle glaucoma (POAG). DESIGN: Prospective, randomized, controlled, multi-surgeon clinical trial. PARTICIPANTS: Enrolled eyes (n = 101) were phakic and had a confirmed POAG diagnosis, normal angle anatomy, mean diurnal intraocular pressure (IOP) 21 to 40 mmHg, and vertical cup-to-disc (C:D) ratio ≤0.9. METHODS: Eyes were randomized (1:1) to receive either 2 stents (iStent trabecular micro-bypass; Glaukos Corporation, San Clemente, CA) or once-daily topical travoprost. MAIN OUTCOME MEASURES: The primary and secondary efficacy end points were the change from screening in mean diurnal IOP at months 12 and 24, respectively, without glaucoma surgery or add-on medication (any medication in stent eyes or a second medication in travoprost eyes). Two additional secondary end points were the proportion of eyes achieving treatment success at months 12 and 24, defined as IOP 6 to 18 mmHg without additional medication or glaucoma surgery. This report shows these efficacy measures through 60 months. Safety measures included best-corrected visual acuity, C:D ratio, visual field, pachymetry, complications, and adverse events. RESULTS: Of 101 enrolled eyes (54 stent eyes, 47 travoprost eyes), 90 eyes (49 stent eyes, 41 travoprost eyes) completed 5-year follow-up. Five-year mean diurnal IOP was 16.5±1.2 mmHg in stent eyes (35.3% reduced vs. 25.5±2.5 mmHg preoperatively; P < 0.0001) and 16.3±1.9 mmHg in travoprost eyes (35.1% reduced vs. 25.1±4.6 mmHg preoperatively; P < 0.0001). During follow-up, add-on medication was initiated in 12 stent eyes (22.2% of the initial 54-eyes) and 18 travoprost eyes (38.3% of the initial 47-eyes). By 5 years, 17% (6/35) of stent eyes and 44% (14/32) of travoprost eyes needed add-on medication to control IOP (P = 0.017). Treatment success was achieved in 77% (27/35) of stent eyes and 53% (17/32) of travoprost eyes (P = 0.04). Both groups exhibited excellent safety. CONCLUSIONS: This prospective randomized trial demonstrates 5-year effectiveness and safety of 2 trabecular bypass stents in patients with newly diagnosed, treatment-naive POAG, with comparably favorable outcomes as topical prostaglandin.

A comparative study on different Descemet membrane endothelial keratoplasty graft preparation techniques.

PURPOSE: To compare different Descemet membrane endothelial keratoplasty (DMEK) graft preparation methods. METHODS: Stripping from the trabecular meshwork (M1) using epithelial spatula; stripping by scoring the peripheral endothelium (M2) using Sinskey hook; stripping by punch method (M3) using donor trephine; Submerged hydro-separation (M4); and pneumatic dissection method (M5) were evaluated. Preparation time, costs, endothelial cell loss (ECL) postpreparation, cell death and morphology were compared. Hoechst/Ethidium/Calcien AM (HEC) staining and Zonula Occludens-1 (ZO-1) expression were analysed. Statistical analysis was performed using one-way anova and; Tukey as post hoc test. RESULTS: A total of 35 corneas (seven per group) were used. Endothelial cell loss (ECL) represented as Mean (SD), in M1, M2, M3, M4 and M5 was 2.7 (5.0), 3.0 (7.4), 1.2 (7.4), 3.3 (7.3) and 4.1 (7.1)%, respectively not showing any difference between the groups (p = 0.96). A significantly higher cell death (p < 0.05) was observed in M4 and M5 compared with M1, M2 and M3. Graft preparation time was significantly shorter in M4 and M5 and longest in M3 (p < 0.05). M3 was the most expensive preparation technique. Minimum pleomorphic cells were observed in M1, M2 and M3, whereas moderate pleomorphism was seen in M4 and M5. Hoechst, Ethidium homodimer and Calcein AM (HEC) staining showed high Ethidium positivity (dead cells) in M4 and M5 with minimum positivity in M1, M2 and M3. Zonula Occludens-1 (ZO-1) was expressed in all the conditions except the denuded areas. CONCLUSION: Graft preparation using Sinskey hook (M2) and donor punch (M3) are reliable methods in terms of efficiency and quality with acceptable range of ECL. The preparation time and associated costs could be a limitation for M3.

Restoration of Aqueous Humor Outflow Following Transplantation of iPSC-Derived Trabecular Meshwork Cells in a Transgenic Mouse Model of Glaucoma.

Purpose: Primary open-angle glaucoma (POAG) is particularly common in older individuals and associated with pathologic degeneration of the trabecular meshwork (TM). We have shown previously that transplantation of induced pluripotent stem cell (iPSC) derived TM cells restores aqueous humor dynamics in young transgenic mice expressing a pathogenic form of human myocilin (Tg-MYOCY437H). This study was designed to determine if this approach is feasible in older mice with more pronounced TM dysfunction. Methods: Mouse iPSC were differentiated toward a TM cell phenotype (iPSC-TM) and injected into the anterior chamber of 6-month-old Tg-MYOCY437H or control mice. IOP and aqueous humor outflow facility were recorded for up to 3 months. Transmission electron microscopy, Western blot, and immunohistochemistry were performed to analyze TM morphology, quantify endoplasmic reticulum (ER) stress, and assess TM cellularity. Results: A 12 weeks after transplantation, IOP in iPSC-TM recipients was statistically lower and outflow facility was significantly improved compared to untreated controls. The number of endogenous TM cells increased significantly in iPSC-TM recipients along with the appearance of TM cells immmunopositive for a marker of cellular division. Morphologically, transplantation of iPSC-TM preserves ER structure 12 weeks after transplantation. However, myocilin and calnexin expression levels remain elevated in transplanted eyes of these 9-month-old Tg-MYOCY437H mice, indicating that ER stress persists within the TM. Conclusions: Transplantation of iPSC-TM can restore IOP and outflow facility in aged Tg-MYOCY437H mice. This type of stem cell-based therapy is a promising possibility for restoration of IOP control in some glaucoma patients.

The Potential of Human Stem Cells for the Study and Treatment of Glaucoma.

PURPOSE: Currently, the only available and approved treatments for glaucoma are various pharmacologic, laser-based, and surgical procedures that lower IOP. Although these treatments can be effective, they are not always sufficient, and they cannot restore vision that has already been lost. The goal of this review is to briefly assess current developments in the application of stem cell biology to the study and treatment of glaucoma and other forms of optic neuropathy. METHODS: A combined literature review and summary of the glaucoma-related discussion at the 2015 "Sight Restoration Through Stem Cell Therapy" meeting that was sponsored by the Ocular Research Symposia Foundation (ORSF). RESULTS: Ongoing advancements in basic and eye-related developmental biology have enabled researchers to direct murine and human stem cells along specific developmental paths and to differentiate them into a variety of ocular cell types of interest. The most advanced of these efforts involve the differentiation of stem cells into retinal pigment epithelial cells, work that has led to the initiation of several human trials. More related to the glaucoma field, there have been recent advances in developing protocols for differentiation of stem cells into trabecular meshwork and retinal ganglion cells. Additionally, efforts are being made to generate stem cell-derived cells that can be used to secrete neuroprotective factors. CONCLUSIONS: Advancing stem cell technology provides opportunities to improve our understanding of glaucoma-related biology and develop models for drug development, and offers the possibility of cell-based therapies to restore sight to patients who have already lost vision.

[An experimental study on mechanism of intraocular pressure decline after allogeneic transplantation of trabecular meshwork].

OBJECTIVE: To study the mechanism of intraocular pressure (IOP) decline after allogeneic transplantation of trabecular meshwork (TM) in primate experimental glaucoma. METHODS: The experimental open-angle glaucoma models were created in 8 rhesus monkey eyes by repeated pangoniophotocoagulation on functional TM using argon laser. After 2, 3, 5 months respectively, avidin-gold or self-erythrocytes, which acted as tracers of the aqueous humor outflow, were perfused into the anterior chamber under normal pressure and volume conditions. 30 minutes later, 2.5% glutaraldehyde was perfused into the anterior chamber under the same conditions. Allograft TMs and photocoagulated area (serving as the control) were observed by light, transmission, scanning electron microscopy and ultrahistochemistry study. RESULTS: The IOP of 5 eyes of 6 experimental eyes became normal. A blood reflux into Schlemm's canal was seen by gonioscopy in 4 eyes. The surface of TMs from graft showed the normal uveal TM cords and intertrabecular spaces. Erythrocytes were found in the intertrabecular spaces and Schlemm's canal. Avidin-gold and vacuolar containing gold were observed in the cells of TM and Schlemm's canal. The TMs of photocoagulated area appeared to be fewer and narrower intertrabecular spaces. The uveal cords of this zone were confused. The content of acid mucopolysaccharides in the grafts was closer to normal than that in the photocoagulated areas. CONCLUSION: TM of allogeneic transplantation not only can survive but also function as an aqueous humor outflow pathway. We consider that this operative procedure can be used for treatment of some cases with glaucoma cautiously.

[Transplantation of functional trabecular meshwork in 9 cases].

On the basis of experiments on rabbits, transplantation of functional trabecular meshwork was performed in 9 cases of absolute angle-closure glaucoma. The surgical reactions were not serious, and the postoperative intraocular pressure decreased significantly by 50%-80% on discharge from the hospital, and in 4 cases followed up for 3 months, by 20%-60%, with 1 case of ocular hypotension. This modality of trabeculoplasty opens a new prospect of ophthalmological organ transplantation for probation.