Streamlining stem cells to treat macular degeneration

Stem cell treatment and biology as a multicellular embryonic concept or adult organismas a symbol for cellular therapy as a 3D illustration.

As we age, so do our eyes; most commonly, this involves changes to our vision and new glasses, but there are more severe 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 details. The result is a blurriness in the central part of our visual field.

The macula is part of the eye’s retina, which is the light-sensitive tissue mostly composed 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 light absorption, cleaning up cellular waste, and keeping the other cells of 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.

Scientist have proposed several methods for converting 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 can take up to a year. And yet, scientists are not clear as to what exactly happens over that period of time.

Mixed cell populations

“None of the differentiation protocols proposed for clinical trials have been scrutinized over time at the single-cell level — we know they can make retinal pigment cells, but how cells evolve to that state remains a mystery,” says Dr Gioele La Manno, a researcher with EPFL’s Life Sciences Independent Research (ELISIR) program.

“Overall, the field has been so focused on the product of differentiation, that the path undertaken has been sometimes overlooked,” he adds. “For the field to move forward, it is important to understand aspects of the dynamics of what happens in these protocols. The path to maturity could be as important as the end state, for example for the safety of treatment or for improving cell purity and reducing production time.”

Tracking stem cells as they grow into RPE cells

La Manno has now led a study with Professor Fredrik Lanner at the Karolinska Institute (Sweden) profiling 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 efficient pluripotent stem cell-based 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 the average expression of RNAs from large populations of cells,” says Alex Lederer, a doctoral student at EPFL and one of the study’s lead authors. “In mixed-cell populations, these measurements may obscure critical differences between individual cells that are important for knowing if the process is unfolding correctly.” Instead, the researchers used a technique called single-cell RNA sequencing (scRNA-seq), which can detect all the active genes in an individual cell at a given time.

Looking 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 out all the 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, thus preventing the formation of contaminant cell populations. “The aim is to prevent mixed cell populations at the time of transplantation, and to make sure the cells at the endpoint are similar to original RPE cells from a patient’s eye,” says Lederer.

What they found was that on the way to becoming RPE cells, stem cells go through a process very similar to early embryonic development. During this, the cell culture took up a “rostral embryo patterning,” the process that develops the embryo’s neural tube, which will go on to become 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: transplanting RPE cells in an animal model

But the point of the differentiation protocol is to generate a pure population of RPE cells that can be implanted in patients’ retinas 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 white albino rabbits, which are what scientists in the field refer to as a “large-eyed animal model.” The operation was carried out following approval by the Northern Stockholm Animal Experimental Ethics Committee.

The work showed that the protocol not only produces a pure RPE cell population but that those cells can continue maturing even after they have been transplanted in the subretinal space. “Our work shows that the differentiation protocol can develop safe and efficient pluripotent stem cell-based therapies for age-related macular degeneration,” says Dr Fredrik Lanner, who is currently making sure the protocol can be soon used in clinics.

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RESTORING LOST VISION New Stem Cell Treatment Developed for AMD

Macular Degeneration Vector Medical Diagram with Eye Anatomy Internal Structure Illustration and Eyesight Disease Types Explanation. Age Related Eyesight Problem, Vision Loss Chart on White Background

Dr. Kapil Bharti of NEI is developing a stem cell-based therapy to prevent and restore vision loss caused by age-related macular degeneration (AMD). He spoke as part of the Sayer Vision Research Lecture Series, with his talk “Translating human RPE biology into disease treatments using induced pluri-potent stem cells.”

AMD typically occurs in individuals over age 65 and targets the retinal pigment epithelium (RPE) of the eye, which forms a monolayer of cells in the back of the eye and provides a secure base for light-sensing photoreceptors to grow on. There are two types of AMD—dry and wet—and Bharti’s new treatment applies to the dry form, which is more common.

In dry AMD, the cells that make up the RPE die off. Without the RPE to support them, photoreceptors also begin to die off, causing vision impairment.

“Symptoms of AMD start off as spotty vision in the center of your vision,” Bharti said, and progresses to the point where patients “lose a big chunk of their central vision and go blind in the center part of their vision.” It is estimated that 1 in 4 individuals over age 80 have some degree of AMD.

Before Bharti’s new stem cell-based therapy of transplanting a patch of RPE cells, AMD patients were treated by injecting new RPE cells in fluid suspension into the retina. This method was not ideal, because the evidence that cells assembled into a functional monolayer is limiting.

Bharti’s research has revealed a method to replace the RPE and its surrounding tissue: using induced pluripotent stem cells (iPS) that are made from the patient’s own blood cells. Naturally occurring pluripotent stem cells are found in embryos and differentiate into the numerous different types of cells in the human body as the embryo develops.

“iPS cells for all practical purposes are identical to embryonic stem cells,” Bharti said, “but the beauty is that we can make them in a dish.” The technique is fairly new but is often preferable for several reasons, such as to avoid the controversy surrounding embryonic stem cell use. iPS cells are also preferable to embryonic stem cells because they are sourced from the patient’s own body and therefore their derivatives are less likely to be rejected when transplanted back into patients.

Bharti and his team take blood cells from AMD patients, reprogram those cells into iPS cells and then give the iPS cells instructions to develop into RPE cells. To make a patch of RPE cells, Bharti and his team let RPE cells mature on a biodegradable scaffold. This entire process takes about 6 months to make fully mature RPE cells starting from patients’ own blood cells.

When it comes time for the new cells to be surgically transplanted into the patient’s eye, what remains of the biodegradable scaffold is implanted along with the cells, to provide structure as the cells integrate with the rest of the retina and grow on their own. Bharti has observed this newly grown layer “halt and reverse progression of disease.”

One of Bharti’s main hopes for this phase 1 trial is “to demonstrate that one can do clinical trials for patients using their own iPS cells, and then we can demonstrate that the patients’ own RPE patch can be delivered safely to the back of the eye, can stay and integrate safely to the back of the eye, and hopefully start functioning over time.”

In the future, he hopes to develop a technique to replace the entire retina, which will help patients with severe dry AMD that have little to no remaining RPE and photoreceptors.

Towards this aim, Bharti’s team also signals some cells to differentiate into photoreceptor and choroidal cells (a structure just behind the RPE) to make a fully functional 3-D-patch. The process is a slow one; component cells are 3-D printed as “bio-ink” onto a biodegradable scaffold, and these cells grow together to form a functional RPE with surrounding tissue as the scaffold breaks down.

View the entire lecture at is external).


First U.S. clinical trial of autologous stem cell therapy to replace dying cells in retina.

Researchers at the National Eye Institute (NEI) are launching a clinical trial to test the safety of a novel patient-specific stem cell-based therapy to treat geographic atrophy, the advanced “dry” form of age-related macular degeneration (AMD), a leading cause of vision loss among people age 65 and older. The geographic atrophy form of AMD currently has no treatment.

For more information on this clinical trial, please click the link below:

NIH launches first U.S. clinical trial of patient-derived stem cell therapy to replace dying cells in retina | National Institutes of Health (NIH)

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