As we age, so do our eyes; most often this involves changes to our vision and new glasses, but there are more serious forms of age-related eye problems. One of them is age-related macular degeneration, which affects the macula -; the back part of the eye which gives us sharp vision and the ability to distinguish details. The result is a blur in the central part of our visual field.
The macula is part of the retina of the eye, which is the photosensitive tissue composed mainly of the eye’s visual cells: cone and rod photoreceptor cells. The retina also contains a layer called the retinal pigment epithelium (RPE), which has several important functions, including absorbing light, cleaning up cellular waste, and keeping other cells in the eye healthy.
The cells of the RPE also nourish and maintain the photoreceptor cells of the eye, which is why one of the most promising treatment strategies for age-related macular degeneration is to replace the aging and degenerated cells of EPR with new products from human embryonic stem cells.
Scientists have proposed several methods to convert stem cells into RPE, but there are still gaps in our knowledge of how cells respond to these stimuli over time. For example, some protocols take a few months while others can take up to a year. And yet, scientists don’t know exactly what happens during this time.
Mixed cell populations
None of the differentiation protocols proposed for clinical trials have been examined over time at the single cell level – we know they can make retinal pigment cells, but how cells evolve to this state remains a question. mystery.
Dr Gioele La Manno, researcher in the Independent Life Sciences Research Program (ELISIR) at EPFL
“Overall, the field has been so focused on the product of differentiation, that the road traveled has sometimes been overlooked,” he adds. “For the field to progress, it is important to understand the dynamic aspects of what is happening in these protocols. The path to maturity could be as important as the end state, for example for processing safety or for improving cell purity and reducing production time.”
Tracking stem cells as they develop into RPE cells
La Manno has now conducted a study with Professor Fredrik Lanner at the Karolinska Institute (Sweden) describing a protocol for differentiating human embryonic stem cells into RPE cells that is actually intended for clinical use. Their work shows that the protocol can develop safe and effective pluripotent stem cell therapies for age-related macular degeneration. The study is published and featured on this month’s cover of the journal Stem Cell Reports.
“Standard methods such as quantitative PCR and bulk RNA-seq capture average RNA expression from large populations of cells,” says Alex Lederer, EPFL PhD student and one of the lead authors of the study. “In mixed cell populations, these measurements can obscure critical differences between individual cells that are important in knowing whether the process is proceeding correctly.” Instead, the researchers used a technique called single-cell RNA sequencing (scRNA-seq), which can detect all active genes in an individual cell at any given time.
Look at intermediate states
Using scRNA-seq, the researchers were able to study the entire gene expression profile of individual human embryonic stem cells throughout the differentiation protocol, which takes a total of sixty days. This allowed them to map all of the transient states within a population as they transformed into retinal pigment cells, but also to optimize the protocol and suppress the growth of non-RPE cells, thereby preventing the formation of populations of contaminating cells. “The goal is to prevent mixed cell populations at the time of transplantation and to ensure that endpoint cells are similar to the original RPE cells from a patient’s eye,” says Lederer.
What they discovered is that to become RPE cells, stem cells go through a process very similar to early embryonic development. During this, cell culture took over a “rostral embryo model”, the process that develops the embryo’s neural tube, which will become its brain and its sensory systems for vision, hearing and taste. After this patterning, the stem cells began to mature into RPE cells.
Eye-to-eye: transplantation of RPE cells in an animal model
But the goal of the differentiation protocol is to generate a pure population of RPE cells that can be implanted into the retina of patients to slow down macular degeneration. So the team transplanted their population of cells that had been monitored with scRNA-seq into the subretinal space of two female New Zealand albino white rabbits, what scientists in the field call a “large-scale animal model.” eyes”. The operation was carried out following the approval of the Ethics Committee for Animal Experiments in North Stockholm.
The work showed that the protocol not only produces a population of pure RPE cells, but that these cells can continue to mature even after being transplanted into the subretinal space. “Our work shows that the differentiation protocol can develop safe and effective pluripotent stem cell-based therapies for age-related macular degeneration,” says Dr. Fredrik Lanner, who is currently ensuring the protocol can be soon used in clinics.
Federal Institute of Technology in Lausanne
Petrus-Reuer, S., et al. (2022) Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation. Stem Cell Reports. doi.org/10.1016/j.stemcr.2022.05.005.