Ophthalmology

Since the generation of induced pluripotent stem cells (iPSCs) in 2006^1,2, they have been studied for regenerative medicine purposes, or cell and gene therapies (CGT) in ophthalmology. In a world first, in 2014, Dr. Masayo Takahashi, transplanted iPSCs derived from skin fibroblasts and differentiated into retinal pigment epithelial (RPE) cells, into a patient suffering from non-neovascular (also known as wet-type) age-related macular degeneration (AMD)³. The RPE cells were formed into a sheet of cells prior to surgery and were able to prevent further deterioration of the patient’s eyesight. This first transplantation was autologous, but later the technology has been expanded to include allogeneic transplants too.

These transplants demonstrate that iPSCs can indeed fulfill the promise of cell therapy to cure severe ophthalmological diseases. The diseases most studied for these advanced therapy medical products (ATMPs) are non-neovascular AMD, retinitis pigmentosa (RP), and Stargardt’s disease (STGD)⁴. But also, glaucoma, cataracts, and corneal cell malfunctioning, including limbal stem cell deficiency (LSCD), attract efforts to generate ATMP-based cures^5–8.

Another source of cells for replacement therapy are the limbal stem cells (LSCs) or corneal stem cells, found in the corneal limbus. Replacement therapy using limbal grafts from donors is increasingly being tested with corneal cell sheets derived from allogeneic LSC samples. In fact, the only stem cell treatment in the eye approved by the European Medicines Agency today is Holoclar® to treat corneal disorders⁹. However, LSCs are a limited source of cells, a hurdle iPSC-based therapies can overcome.

iPSCs to Treat Blindness caused by LSCD?
LSCD is the cause of corneal blindness for over six million people worldwide⁸. Here, simply replacing the corneal sheet is not enough: The entire limbal niche, including the limbal scaffold, microenvironment and the LSCs themselves, is crucial to maintain a healthy pool of LSCs which can continuously proliferate and differentiate into the corneal sheet for full ocular surface restoration.

To treat a degenerative eye disease such as AMD, a suspension of retinal pigment epithelium (RPE) cells derived from iPSCs are injected into the subretinal space directly, or they are transplanted on a scaffold mimicking Bruch’s membrane for support^10,11. These two types of ATMPs differ significantly in their final format and formulation, but common to them is that tight regulation provides precise control of the cell differentiation and maintenance formulation.

Efforts to mimic Bruch’s membrane (or the vitreous lamina), means intense investigation into scaffold materials and structures for the RPEs to attach to, and use for transplantation in AMD patients where that compartment is damaged.

Also, production scales differ tremendously whether employing an autologous or allogeneic therapeutic approach and comes with challenges of their own. High cost due to single sample-production of autologous regenerative tissues make them less favorable than the low-cost multiple sample-production of allogeneic tissues. But at the same time, the risk of allogeneic transplant rejection means using immunosuppressants, which in turn can have negative outcomes for patient health and disease management¹¹.

Scalable Cell Counting Technology for Successful Therapy Formulation

Whether working on challenging iPSC differentiation protocols to obtain pure RPE cell populations, optimizing the scaffold to best suit the retinal or limbal niche, upscaling the production volumes for the therapeutic cells or formulating the final product – or something entirely different – it is central to quantify and qualify the cell population at multiple steps of the production pipeline and within multiple departments.

To ensure consistency and reproducibility in all steps of the product development and formulation, having a precise method to estimate cell concentration and viability is key. The NucleoCounter^® NC-202™ automated cell counter offers specialized assays for cells in single-cell suspension, in aggregates, and grown on microcarriers or other scaffolds. It is easy to use, eliminates human interference and variation from the counting process and takes only 30 seconds per cell count.

With GMP/ 21 CFR Part 11-compliant NC-View™ software, the NucleoCounter® NC-202™ is scalable and can be used from the earliest R&D protocols, through process development, manufacturing process monitoring, and QC validation steps of the final formulation. The NC-202™ is a robust cell counter for effortless integration across SOPs, operators, and departments, ensuring reliable data comparison and validation.

1. K Takahashi and S Yamanaka: Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006; 126, 663–676. 
2. K Takahashi, K Tanabe, M Ohnuki, et al.: Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007; 131, 861–872. 
3. M Mandai, A Watanabe, Y Kurimoto, et al.: Autologous induced stem-cell-derived retinal cells for macular degeneration. N. Engl. J. Med. 2017; 376, pp. 1038-1046. 
4. Ramsden CM, Powner MB, Carr AJF, et al. Stem cells in retinal regeneration: past, present and future. Development 2013; 140:2576-2585. 
5. P Gain, R Jullienne, Z He, et al.: Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol. 2016; 134(2): 167-173. 
6. F Yuan, M Wang, K Jin, et al.: Advances in Regeneration of Retinal Ganglion Cells and Optic Nerves. Int. J. Mol. Sci. 2021; 22, 4616. 
7. M Parekh, V Romano, K Hassanin, et al.: Biomaterials for corneal endothelial cell culture and tissue engineering. J Tissue Eng. 2021; 12: 1–19. 
8. ÖB Selver, A Yağcı, S Eğrilmez, et al.: Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation. Turk J Ophthalmol. 2017; 47(5): 285-291. 
9. G Pellegrini, D Ardigò, G Milazzo, et al.: Navigating Market Authorization: The Path Holoclar Took to Become the First Stem Cell Product Approved in the European Union. Stem Cells Transl Med. 2018; 7(1): 146–154. 
10. P Fernández-Robredo, A Sancho, S Johnen, et al.: Current Treatment Limitations in Age-Related Macular Degeneration and Future Approaches Based on Cell Therapy and Tissue Engineering. 2014; ID:510285. J Ophthalmol. Special edition: Age Macular Degeneration: Etiology, Prevention, Individualized Therapies, Cell Therapy, and Tissue Engineering.  
11. S Khateb, S Jha, K Bharti, et al.: Cell-Based Therapies for Age-Related Macular Degeneration. Adv Exp Med Biol. 2021; 1256: 265-293. 
12. National Eye Institute: https://www.nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/cataract-data-and-statistics. 
13. Medline: https://medlineplus.gov/genetics/condition/retinitis-pigmentosa/.