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Genetic Linkage

Retinal Stem Cells and Eye of Newt

Human RPE cells in culture look like cobblestones; 3% of them behave like stem cells, in dishes. Can they treat eye diseases from within? (Tim Blenkinsop)
More than a decade before Sally Temple, PhD, and her husband Jeffrey Stern, MD, PhD, discovered stem cells in human eyes, they suspected the cells would be there. They knew it from the salamanders.

When William Shakespeare included “eye of newt” ingredients of the Three Witches’ brew in Macbeth, he probably knew what he was doing. Dr. Temple, who grew up in northern England, said it’s long been common knowledge there that newts can regrow their parts. In the late 1800s, biologists began to study regeneration in salamanders.

By the 1950s, embryologists had discovered that certain amphibian eyes regenerate thanks to a single layer of cells, called the retinal pigment epithelium (RPE), which hugs the photoreceptors (the rods and cones). The RPE is the source of the new stem cells -- RPESCs – that are housed in a spectacular building overlooking the Hudson River in Rensselaer, New York. Here, Drs. Temple and Stern and their two dozen associates form the Neural Stem Cell Institute/Regenerative Research Foundation.

Amphibians became an essential part of developmental biology. In my lab in grad school at Indiana University, when we’d tire of counting flies, we’d fish out newly-fertilized eggs from the tank of Sally (not Temple) and Gerry. They were our pet axolotls, rescued from the salamander colony upstairs. I loved watching the cells cleave, forming soccer-ball-like early embryos right before our eyes.

Some of the earliest "nuclear transplantation" (aka cloning) experiments in animals used amphibians, starting in the 1950s. And some of the most elegant experiments in biology were those of Sir John Gurdon, who shared the Nobel Prize in Physiology or Medicine in 2012 with Shinya Yamanaka (of more recent induced pluripotent stem cell [iPS]) fame).

Dr. Gurdon cloned adult Xenopus laevis frogs from tadpole cells of the African clawed frog, a species once noted for use in a pregnancy test for people. And he was one of Dr. Temple’s professors at Cambridge, where she did her undergraduate work, “not realizing I was in a hotbed of developmental biology,” she recalled.

With this legacy, by the time Dolly the cloned sheep hit the headlines in 1996 and people panicked at the specter of mass-produced Nazis and dinosaurs, developmental biologists wondered, WHAT TOOK SO LONG?

Among the amphibians, salamanders are especially adept at regenerating their eyes, which have a layered structure similar to our own. “If they have retinal damage, or you remove the retina, the RPE is activated. It's cells divide, differentiate, and make a new retina,” said Dr. Temple. That’s a classic description of a stem cell. “We knew these cells could behave like stem cells and regenerate the retina. It’s locked inside the cells and if we can figure out how to unlock it, we can make not just RPE, but photoreceptors.” She paused a moment. “Regeneration is THE question in science and medicine, isn’t it?”

The RPE is an oft-underappreciated cell type, in a similar situation to the neuroglia that keep neurons alive. Like Anne Hathaway’s “supporting” but critical role in Les Miserables, the thin RPE is essential for the functioning of the intricately folded, majestic rods and cones, and therefore, for sight.

The importance of the RPE makes it all the most astonishing that it forms when we’re one-month-old embryos and stays put, its cells never dividing unless something goes wrong.

“We have embryonic cells in our eyes, but they’re not embryonic stem or pluripotent cells. They’re restricted to making a few cell types,” Dr. Temple said as she sketched cells, something she does nearly continually when talking about her work.

The RPE stem cells aren’t totipotent, as only fertilized ova can be, nor are they pluripotent, like embryonic stem cells or iPS cells. They’re multipotent, facing only a few fates.

The researchers obtained human RPEs from eye banks that discard what remains after harvesting corneas. In a body, RPE cells are quiescent, but in culture they divide like crazy, forming coatings of cobblestone-like cells festooned with RPE markers.

To show that an RPE cell could be a stem cell, the researchers gently removed one at a time and transferred it to its own dish, giving it space to divide. This would reveal the defining characteristic of a stem cell: the ability to self-renew, to copy itself. (If all a stem cell could do was “turn into any cell type,” as they’re often defined, the culture would soon poop out.)

Most of the rehomed RPE cells promptly perished, but about 3% of them self-renewed and yielded RPE cells. One original isolated RPE cell with this hidden talent could, theoretically, generate a thousand RPE cells.

A few weeks ago, Tim Blenkinsop, PhD, showed me a plate of RPE cells at the Neural Stem Cell Institute. They all looked the same under the microscope. Was I missing something? “We don’t know which are which. The stem cells self-renew. Remove one and it can re-establish a monolayer. But there are no pure cultures of RPE-SC,“ he explained.

Dr. Temple continued. “This is a tissue that wasn’t previously understood to have a stem cell line. It isn’t a proliferative tissue, but hidden within it is this population of cells that can be activated to divide.” Because the RPE yields stem cells in a dish, chances are they can in an eyeball too, perhaps in response to injury or illness.

Drs. Stern and Temple see their cells as “another horse in the race,” alongside human ES cells, which are providing RPE to treat Stargardt’s macular dystrophy and macular degeneration. Another approach is to generate RPE from iPS cells. At the Neural Stem Cell Institute, Barb Corneo, PhD, and her husband, Dr. Blenkinsop, are “the keepers of the cells,” including iPS cells.

“The iPS-cell-derived RPE stem cells are more pigmented,” Dr. Corneo said “and the adult ones are lighter” added Dr. Blenkinsop, in that complete-each-other’s-sentences way I’ve encountered before in spouses who share science. The adult-derived ones dilute their pigment when they divide.

To observe the cells I wore a blue lab coat and purple gloves so I didn’t sicken the cells. Dr. Corneo slid a culture dish harboring cells from various eye parts under the microscope. “We use these as donors to make iPS cells,” she said, replacing the dish with a 6-welled container. “These are just plain iPS cells, the island among the feeder cells,” she explained.

Like a chocolate lover seeing a box of Godivas, I recognized the iPS cells instantly, having seen them in so many images since their invention in late 2007. It was as thrilling as watching salamander embryo cells cleave.

“Those cells can make anything. Barb just pushes them down the pathway to make RPE,” Dr. Temple explained.

The RPESCs are of intense interest because of what they can become. When the researchers exposed RPE cells in culture to a cocktail of factors promoting specialization as neurons, the cells become marked with molecules characteristic of nerve cells – suggesting that Dr. Temple’s dream of regenerating rods and cones may be possible.

The goal is to use the cells to treat eye diseases. And they seem perfect. “Embryonic stem cells very rapidly form RPE by default. RPE is one of the first tissues to terminally differentiate in normal development so in a way our cell is an intermediate cell. Rather than control the early steps, which we don’t really understand, why don’t we start later, closer to our target tissue? Rather than a pluripotent cell that can do everything, we have a stem cell that can do what we want. It’s nature’s specialist,” explained Dr. Stern, an ophthalmologist.

Many of Dr. Stern’s patients have age-related macular degeneration (AMD), as do 12-15 million people in the U.S. and 25% of people over 60. Central vision fades as rods and cones die. Ten percent of cases are “wet,” in which blood vessels in the eye overextend and leak in response to the RPE forming scar tissue. The RPESCs would treat dry AMD, possibly replacing relocation of part of a patient’s RPE to patch blurry spots, according to Pete Coffey, PhD, director of the London Project to Cure Blindness and professor of cellular therapy and visual sciences at the UCL Institute of Ophthalmology.

“In an ideal world we could take some of these RPE cells from a patient and grow them up. To treat AMD we’d only need 30,000 to 50,000 cells to cover the part that’s degenerated. We want to access the salamander potential for a short period to repair the RPE or the retina,” Dr. Temple said. The cells can also be used as a “disease in a dish” to test drugs.

The stem cell characteristic that frightens critics is what entices many researchers: discovering new fates. The RPESC can be coaxed to give rise to what Dr. Temple calls “bizarre mesenchymal things” – bone, cartilage, and fat.

The two fates – RPE and mesenchyme – hail from different layers of the earliest embryo, the ectoderm and mesoderm, respectively. Practically speaking, the ability of the eye to produce bone, cartilage, and fat explains two strange diseases.

Proliferative vitrioretinopathy (PVR) and phthisis bulbi (“macular pucker”) are medical emergencies. “The RPE moves, proliferates, and makes fibers that pull the retina off. No one had shown previously that RPE could make mesenchymal cells, so when we discovered this potential, ophthalmologists became excited because maybe we could prevent these diseases,” Dr. Temple explained. “In some awful situations, the retina can calcify – where does the bone come from? Maybe from misbehaving RPE,” she added.

So the RPESCs may have a dual use. “One cell under some circumstances can make healthy RPE that can be used in replacement therapy, and in another set of circumstances can push into an abnormal mesenchymal fate that can be a model to screen drugs for PVR and macular pucker,” Dr. Temple said.

I normally wouldn’t write about work published a year ago that’s already been covered quite well, but I have a few updates, ranging from the profound to the personal.

1. December 12, 2012 was an important date for two reasons. The Empire State Stem Cell Board and the New York State Stem Cell Program recommended the Regenerative Research Foundation, the not-for-profit arm of the Rensselaer facility, for a $10.6 million grant. “Our tiny institute got the top score! We were astonished,” said Dr. Temple. The Neural Stem cell Institute formed the Retinal Stem Cell Consortium to bring others in to do some of the preclinical work.

The same day, the National Academy of Sciences released conclusions of a year-long investigation of the progress of the California Institute of Regenerative Medicine, critical of the $6 billion program to fund stem cell research that began in 2004 after voters approved Proposition 71.

2. The RPESC seem to be an entirely new class of stem cells because there isn’t a stain that makes them stand out, as can the better-studied neural stem cells in our brains. (These cells were discovered in rodent brains in 1912; then in birds, who use them to learn songs; then in tree shrews and marmosets; and finally in tongue cancer patients who donated their brains. The entire strange tale is in my little-known book "Discovery: Windows on the Life Sciences.")

3. While I’m glad that the bioethics community seems to have left embryos to tackle stem cell tourism, the embryo issue resurfaced during the Presidential election. It’s nice to know that many researchers are forging ahead with the pools of potential that are normally right in our bodies – sometimes hidden in plain sight.

4. Finally, and more philosophically, is the irony of RPE cells that reinvent themselves as stem cells. Their existence counters the premise of my book "The Forever Fix: Gene Therapy and the Boy Who Saved It".

The book’s central figure, young Corey Haas, had a “forever fix” of his inherited blindness when billions of viruses injected into his eyes delivered replacement genes – and in just days he saw the sun for the first time, at the Philadelphia zoo. Corey is unlikely to need another gene therapy, precisely because his doctored RPE cells are not expected to ever divide, which would dilute the correction. Could RPESCs naturally nestled within his eyes have been reawakened to heal from within, given the right signals?

This is what I love about science: the enigmas and the inconsistencies, the unexpected findings that fuel discovery. That’s why there’s no such thing as “scientific proof,” despite what advertisers pitch to the public. New observations and unanticipated results continually alter what we thought we knew – and entice us to think harder and ask different questions about how nature works.

This blog was simultaneously published at http://blogs.plos.org/dnascience/, which has 10 photos.
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