As we age, so do our eyes; Most commonly, this includes changes in our vision and new glasses, but there are more severe forms of age-related eye problems. One of them is age-related macular degeneration, which affects the macula – the back part of the eye that gives us sharp vision and the ability to discern details. The result is a blurring of the central part of our field of view.
The macula is part of the retina, a light-sensitive tissue composed mostly of the eye’s optical cells: the cone cells and the photoreceptor rod. The retina also contains a layer called the retinal pigment epithelium (RPE), which has many important functions, including absorbing light, cleaning up cellular waste, and maintaining the health of other eye cells.
RPE cells also nourish and maintain ocular photoreceptor cells, which is why one promising treatment strategy for age-related macular degeneration is to replace degraded RPE cells with new cells grown from human embryonic stem cells.
Scientists have proposed several ways to convert stem cells into RPE, but there is still a gap in our knowledge of how cells respond to these stimuli over time. For example, some protocols take a few months while others may take up to a year. However, scientists are not clear about what exactly happens during that time period.
Mixed cell groups
“None of the differentiation protocols proposed for clinical trials has been examined over time at the single-cell level—we know they can make pigment cells in the retina, but how the cells develop into this state remains a mystery,” Joel La Mano, PhD, a researcher in the EPFL Research Program Independent Life Sciences (ELISIR), as reported in a press release.
“Overall, the field has been so focused on the product of differentiation that the path taken has sometimes been overlooked,” he explained in the statement. For the field to move forward, it is important to understand aspects of the dynamics of what is happening in these protocols. The path to maturation may be just as important as the end state, for example for treatment safety or to improve cell purity and reduce production time.”
Trace stem cells as they grow into RPE cells
La Mano has now led a study with Professor Fredrik Lanner at Karolinska Institutet (Sweden) to profile a protocol to differentiate human embryonic stem cells into RPE cells that are in fact intended for clinical use. Their work shows that the protocol can develop safe and effective stem cell-based therapies for age-related macular degeneration. The study was published and featured on the magazine’s cover for this month Stem Cell Reports.
“Standard methods such as quantitative PCR and bulk RNA-seq capture the average expression of RNAs from large populations of cells,” Alex Lederer, a doctoral student at EPFL and one of the study’s lead authors said in a press release. “In mixed cell populations, these measurements may mask critical differences between individual cells that are important for seeing if the process is unfolding properly.”
Instead, the statement noted, the researchers used a technology called single-cell RNA sequencing (scRNA-seq), which can detect all the active genes in an individual cell at a given time.
Consider middle countries
Using scRNA-seq, the researchers were able to study the entire gene expression profile of individual human embryonic stem cells through a differentiation protocol, which takes a total of sixty days. This allowed them to map all transients within a population as they grow into retinal pigment cells, but also to improve the protocol and suppress non-RPE cell growth, thus preventing the formation of staining cell populations. “The goal is to prevent mixed cell pools at the time of transplantation, and to ensure that the cells at the end point are identical to the original RPE cells from the patient’s eye,” Lederer says.
What they found is that on the way to becoming RPE, stem cells go through a process very similar to early embryonic development. During this, the cell cultures took on the “rotal fetal pattern,” the process that develops the fetus’s neural tube, which will become its brain and sensory systems for vision, hearing and taste. After this pattern, stem cells began to mature into RPE cells.
Head to head: RPE cell transplantation in an animal model
But the goal of the differentiation protocol is to generate a pure pool of RPE cells that can be transplanted into the retina of patients to slow macular degeneration. So the team transplanted their populations of cells monitored by scRNA-seq into the subretinal space of two New Zealand white rabbits, which scientists in the field refer to as a “big-eyed animal model.” The operation was carried out after approval by the Experimental Animal Ethics Committee in northern Stockholm.
The work showed that the protocol not only produced a pure RPE cell population but that these cells could continue to mature even after transplantation into the subretinal space.
“Our work shows that the differentiation protocol can develop safe and effective stem cell-based therapies for age-related macular degeneration,” said Frederic Lanner, PhD, who is currently confirming the protocol can soon be used in clinics. Release.
Other contributors include Novo Nordisk A/S and University of California San Francisco.
Sandra Petros Reurier, Alex R. Vilkasca, Helder Andre, Eric Sundstrom, Aparna Bahaduri, Arnold Kriegstein, Anders Quanta, Joel La Mano and Frederic Lanner. Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation. Stem Cell Reports June 14, 2022. DOI: 10.1016/j.stemcr.2022.05.005