Stem cell tightening to treat macular degeneration

As we age, our eyes age too; The most common are vision changes and new glasses, but there are more serious forms of age-related eye problems. One of these is age-related macular degeneration, which affects the macula — the back part of the eye that gives us sharp vision and the ability to distinguish detail. The result is a blur in the central part of our field of vision.

The macula is part of the eye’s retina, the light-sensitive tissue made up primarily of the eye’s photoreceptor cells: cone and rod photoreceptor cells. The retina also contains a layer called the retinal pigment epithelium (RPE) that has several important functions, including absorbing light, cleaning cellular debris, and keeping the other cells in the eye healthy.

The cells of the RPE also nourish and maintain the eye’s photoreceptor cells, which is why one of the most promising treatment strategies for age-related macular degeneration is to replace aging, degenerating RPE cells with new ones grown from human embryonic stem cells.

Scientists have proposed several methods for converting stem cells into RPE, but our understanding of how cells respond to these stimuli over time is still patchy. For example, some logs last a few months while others can last up to a year. And yet it is not clear to scientists what exactly happens during this period.

Mixed cell populations

“None of the differentiation protocols proposed for clinical trials have been studied at the single-cell level over time — we know that they can form retinal pigment cells, but how cells develop into this state remains a mystery,” Gioele La Manno, PhD, a researcher in the EPFL Life Sciences Independent Research (ELISIR) programme, as noted in a press release.

“Overall, the field has become so focused on the product of differentiation that the path taken has sometimes been overlooked,” he explained in the statement. “In order for the field to move forward, it is important to understand aspects of the dynamics of what is happening in these protocols. The route to maturity could be just as important as the final state, for example for treatment safety or improving cell purity and reducing production time.”

Tracking stem cells as they grow into RPE cells

La Manno has now led a study together with Professor Fredrik Lanner at the Karolinska Institute (Sweden) in which a protocol for the differentiation of human embryonic stem cells into RPE cells was created, which is actually intended for clinical use. Their work demonstrates that the protocol can develop safe and efficient pluripotent stem cell-based therapies for age-related macular degeneration. The study will be 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 the average expression of RNAs from large cell populations,” said Alex Lederer, a PhD student at EPFL and one of the study’s lead authors, in the press release. “In mixed-cell populations, these measurements can obscure critical differences between individual cells that are important for knowing whether the process is proceeding properly.”

Instead, the researchers found that the researchers used a technique called single-cell RNA sequencing (scRNA-seq), which can detect all active genes in a single cell at any given time.

consider intermediate states

Using scRNA-seq, the researchers were able to examine the entire gene expression profile of individual human embryonic stem cells during the differentiation protocol, which takes a total of 60 days. This allowed them to map all transient states within a population as they grew into retinal pigment cells, but also to optimize the protocol and suppress the growth of non-RPE cells, preventing the formation of contaminating cell populations. “The goal is to prevent mixed cell populations at the time of transplantation and to ensure that the cells at the endpoint resemble the original RPE cells from a patient’s eye,” says Lederer.

They found that on the way to becoming RPE cells, stem cells go through a process that is very similar to early embryonic development. In doing so, the cell culture recorded a ‘rostral embryo pattern’, the process that develops the embryo’s neural tube, which later becomes its brain and 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 animal models

However, the purpose 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 cell population, which had been monitored with scRNA-seq, into the subretinal space of two female New Zealand albino white rabbits, which scientists in the field call a “big-eyed animal model.” The operation was carried out after approval by the Ethics Committee for Animal Experimentation in north Stockholm.

The work demonstrated that not only does the protocol produce a pure RPE cell population, but that these cells can continue to mature even after transplantation into the subretinal space.

“Our work demonstrates that the differentiation protocol can develop safe and efficient pluripotent stem cell-based therapies for age-related macular degeneration,” said Fredrik Lanner, PhD, who is currently ensuring the protocol can soon be used in clinics, in a news release.

Other contributors are Novo Nordisk A/S and the University of California San Francisco.

Relation

Sandra Petrus-Reurer, Alex R. Lederer, Laura Baqué-Vidal, Iyadh Douagi, Belinda Pannagel, Irina Khven, Monica Aronsson, Hammurabi Bartuma, Magdalena Wagner, Andreas Wron, Paschalis Efstathopoulos, Elham Jaberi, Hanni Willenbrock, Yutaka Shimizu, J. Carlos Villaescusa, André Helder, Erik Sundstrom, Aparna Bhaduri, Arnold Kriegstein, Anders Kvanta, Gioele La Manno, Fredrik Lanner. Molecular profiling of stem cell-derived differentiation of retinal pigment epithelial cells for clinical translation established. Stem Cell Reports June 14, 2022. DOI: 10.1016/j.stemcr.2022.05.005