Seeing Is Believing: Exploring the Mechanisms of Lens Regeneration
The regenerative medicine field is an exciting area of research, because it is promising for the treatment of damaged or nonfunctional organs and tissues. However, because humans have limited regenerative capabilities compared with other species, using this knowledge to alter one of the most delicate structures in the body, such as the eye, is challenging. I am fascinated by the anatomy of the eye, because there are many delicate parts involved in making the simplest actions possible. The eye has limited regenerative capabilities as an organ, but each structure within the eye maintains a subset of regenerative abilities. In humans, eye tissues often endure permanent vision impairment when damaged. Applying regeneration concepts to heal tissues that would otherwise succumb to irreversible damage is what makes studying regeneration so crucial for the future of medicine.
The purpose of my experiment, funded by a Summer Undergraduate Research Fellowship (SURF), was to gain a better understanding of Yamanaka factors and their function in cellular reprogramming and regeneration, with an emphasis on lens regeneration in the newt Pleurodeles waltl (also referred to as the Iberian ribbed newt). Yamanaka factors are a specialized group of proteins (often referred to as transcription factors) whose presence is crucial for reprogramming somatic cells into pluripotent stem cells (Aguirre et al., 2023). The term somatic cell refers to any specialized cell in the body, such as an eye or skin cell. Pluripotent stem cells are an unspecialized, simplified version of cells that can morph into almost any type of specialized cells in the body. For regeneration to occur, the somatic cells in damaged tissue must revert to a stem cell–like state before they can mature into healthy tissue. Yamanaka factors must be present for this to happen.
Through this reprogramming process, tissues can sustain redifferentiation into new specialized tissue. There are several applications of redifferentiation in medicine, such as stem cell therapy and drug screenings (Mayo Clinic, 2024). By learning more about this process, we can develop personalized treatments to generate healthy tissue using patient-specific stem cells. The research I present in this research brief contributes to the field of regenerative medicine by examining the cellular mechanisms behind tissue regeneration. Understanding the fine details of regenerating systems in newts can help us recognize similar mechanisms in areas of the human body such as the eye. In turn, it is hoped that this knowledge can help scientists develop new treatments for human ophthalmic applications.
The Inquisitive Nature of Iberian Ribbed Newts
Like so many other biomedical research areas, the ophthalmology field has become reliant on model organisms that contain structures similar to humans’ but also maintain the ability to regenerate. This is why newts and axolotls (a class of amphibian) are used so frequently. These models allow researchers to dig into the specific cellular mechanisms governing cellular regeneration in an environment that is, ideally, compliant with potential application in the human body. The Iberian ribbed newt is a commonly sought-after model organism for regenerative research because of their ability to reproduce structures that are considered vital for human vision. These structures include (but are not limited to) the lens and retina. This remarkable potential for Pleurodeles waltl is enabled by highly specific gene expression and reprogramming levels within each individual cell (Matsunami et al., 2019).
Various Yamanaka factors are responsible for maintaining strict regulation of gene expression levels, which manipulate cellular function. During lens regeneration in newts, these transcription factors play a crucial role in the reprogramming of iris epithelial cells into lens cells by causing downregulation of iris-specific genes and upregulation of lens-specific genes (Aguirre et al., 2023). Downregulation is when a cell reduces the activity or production of a molecule, while upregulation does the opposite—increasing the activity or production of a molecule. These processes are more commonly observed when too much (downregulation) or not enough (upregulation) of a particular cell signal is present in the surrounding tissue. A cell signal is a molecular message used to trigger a particular response via receptors on the surface of cells. In the context of Yamanaka factors, cell signals are used for pluripotent stem cell generation by upregulating or blocking reprogramming.
For this project, I intended to identify the specific role of several different Yamanaka factors (including Sox2, c-Myc, and others) in cellular reprogramming, and hypothesized that all play a significant role regardless of each one’s unique function. As part of this research, I analyzed several reprogramming inhibitors for their effect on the tissue regeneration process. The purpose of using reprogramming inhibitors is to halt or slow the process of turning typical, specialized cells (like eye, skin, heart, and so on) into stem cells, which are a more flexible form of cells. By blocking the signals and factors required for the reprogramming process, researchers can analyze and then alter regeneration mechanisms, which will in turn allow us to re-create this process where it is normally missing, such as in human tissues. To analyze the reprogramming process, we conducted trials using various inhibitor drug treatments. The inhibitor drugs enhance Yamanaka factors by controlling gene expression and promote changes in signaling pathways that encourage differentiation of cells into different cell types. By studying lens-specific genes in combination with inhibitor data, we receive a more accurate understanding of the regeneration mechanisms.
Methods and Preliminary Results
This research aimed to incorporate a multitude of molecular techniques to cover all the possible effects the reprogramming inhibitors may have on lens regeneration. The experimental design required phylogenetic analysis, tissue imaging, and histology. Phylogenetic trees were constructed and studied to understand the evolutionary journey of Yamanaka factors between Pleurodeles waltl and a variety of other species. Phylogenetic analysis is a supplemental tool that is helpful when looking at the evolutionary history of significant lens-specific genes, especially when considering the genes humans and newts share.
To conduct the inhibitor drug treatment trials, we treated the newts’ housing water with diluted drugs for sixteen days. Individual newts had their own housing and water source, so each newt was subject to one of several drug trials. At the conclusion of this period, the newts were euthanized. Then, the eyes were extracted, separated into tubes based on their respective drug treatment, and dehydrated with ethanol. The dehydration process was followed by several rounds of xylene and paraffin washes. Dehydration is a necessary step in this process to expel water, and ensures the samples are embedded solely in paraffin wax. Once placed in the paraffin, the eyeballs were oriented to prepare for sectioning using a microtone to obtain very thin slices of tissue.
After sectioning was completed, we used staining techniques with hematoxylin and eosin (H&E) to observe the tissues under a microscope and assess inhibition of regeneration. At the time of publication, this is where we stand in the research process. This histological analysis, currently underway, will allow us to see the structural changes associated with the stages of lens regeneration. It will also allow us to determine the specific role of each different Yamanaka factor in cellular reprogramming. Our goal is to continue this project using genetic, molecular, and biochemical approaches focused on the specific Yamanaka factors and inhibitor treatments that yield the most promising results.
From the brief evaluation completed thus far, the results suggest one of the inhibitor drugs I used in my experiment may be effective in halting the reprogramming, and in the subsequent regeneration process in the lenses of newts. However, continuation of this project, along with additional research, needs to be completed to verify this potential finding. If this study is replicated with the same result, this discovery could alter the field of regenerative medicine by providing researchers with an improved method for investigating the specific factors and steps involved in reprogramming. The use of inhibitor drugs to learn more about the regeneration process would ultimately enable researchers to develop certain treatments, such as stem cell therapy. This could also accelerate the drug development process, making a significant contribution to both science and medicine.
Conclusion
Overall, the results of this research may imply crucial information needed for further understanding of regenerative medicine, which aligns with the goal of Dr. Sousounis’s lab—to one day contribute to the development of regenerative therapies for human disease, particularly those involved in vision. As part of this research team, I immersed myself in the scientific process in a way I never have before. This experience provided knowledge of hands-on experiments, data analysis approaches, and theoretical applications for real-world problems. I gained a deeper appreciation for precise data collection, the fine details of experimental design, and the importance of proper interpretation of results. In addition, I found a passion for regenerative biology and its potential to make an impact on the medical field. I am excited to present my research at the Undergraduate Research Conference (URC) this spring and to continue pursuing research opportunities after graduation.
I would like to thank my donor, Mr. Dana Hamel, for funding my research, and my mentor, Konstantinos Sousounis, for his support and confidence in me to succeed. In addition, I’d like to express my gratitude for the other members of Dr. Sousounis’s lab (especially Olivia Williams and Kelsey Ahearn), who constantly inspire me to improve my research skills. Without the Hamel Center for Undergraduate Research or the Sousounis lab, I would not have received this opportunity.
References
Aguirre, M., Escobar, M., Forero Amézquita, S., Cubillos, D., Rincón, C., Vanegas, P., Tarazona, M. P., Atuesta Escobar, S., Blanco, J. C., & Celis, L. G. (2023, August 26). Application of the Yamanaka transcription factors Oct4, Sox2, Klf4, and c-Myc from the laboratory to the clinic. Genes 14(9), 1697. https://doi.org/10.3390/genes14091697
Matsunami, M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T., Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., Takeuchi, T., Suzuki, K.-I. T., Agata, K., Shigenobu, S., & Hayashi, T. (2019, June 1). A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes. https://pmc.ncbi.nlm.nih.gov/articles/PMC6589553/
Mayo Foundation for Medical Education and Research. (2024, March 23). Stem cells: What they are and what they do. Mayo Clinic. https://www.mayoclinic.org/tests-procedures/bone-marrow-transplant/in-depth/stem-cells/art-20048117
Author and Mentor Bios
Jenna Loporcaro is a biochemistry, molecular and cellular biology major from Rochester, New Hampshire, who will graduate from the University of New Hampshire (UNH) in May 2025. She received a Summer Undergraduate Research Fellowship (SURF) in 2024 to complete the work described in this research brief. In addition to being a student ambassador with the Hamel Center for Undergraduate Research, she is a member of Partners for World Health at UNH and the Beta Kappa chapter of Sigma Alpha sorority. Jenna plans to pursue medicine and further research endeavors after graduation.
Konstantinos Sousounis is an assistant professor in the Department of Molecular, Cellular and Biomedical Sciences at UNH. His research focuses on how certain vertebrate animals, like newts and axolotls, are capable of regenerating tissues and organs, and how to induce regeneration in incompetent animals/tissues.
Copyright 2025 © Jenna Loporcaro
