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Patient-Specific Stem Cells Recapitulate Age-Related Macular Degeneration

Age-related macular degeneration obliterates the visual field, from the center outwards. (Natl Eye Inst)
Age-related macular degeneration obliterates the visual field, from the center outwards. (Natl Eye Inst)

Stem cell debate and hype continue, with each advance distancing the field from embryos while promising replacement parts as stem cells “turn into” everything from hearts to gizzards. Meanwhile, many researchers have been quietly pursuing the immediate promise of the cells – conjuring the beginnings of diseases in dishes. DNA Science featured “brain organoids” last summer, before the world disappeared beneath a glacier.

Induced pluripotent stem (iPS) cells, more commonly called reprogrammed cells, start out as skin or other somatic (body) cells, zip back in developmental time to a stem-cell-like state, then are coaxed to assume whatever guise researchers wish to study. The cells are the route to personalized implants, because they come from the patient who needs a spare part. But that will require a lot of testing. More immediate, and more exciting I think, is when iPS cells serve as living time machines.

Imagine taking an affected cell from a person very sick from a degenerative disease, and reversing the clock, glimpsing in a lab dish how things began to go wrong.

A good disease to dissect using reprogrammed cells is age-related macular degeneration (AMD), a form of encroaching blindness that affects 12 to 15 million people in the U.S. and a quarter of those over 60. Incidence will double as the population ages over the next decade. In AMD the visual field becomes wavy, faded, and blocked, from the center outwards, greatly interfering with daily life.

iPS cells (Stephen Tsang and colleagues, Columbia University)
iPS cells (Stephen Tsang and colleagues, Columbia University)

AMD is a great candidate to mirror in reprogrammed cells because iPS cells left alone and not given biochemicals to steer their specialization will, for reasons unknown, eventually become the very tissue responsible for the gradual visual loss – the retinal pigment epithelium, or RPE. A DNA Science post from last year describes the RPE in detail.

Like many diseases, AMD arises from an interplay of environmental and genetic influences. A team led by Stephen Tsang, an ophthalmologist and geneticist at the Harkness Eye Institute at Columbia University, used iPS cells to reveal the gene-environment interaction that underlies AMD, with a practical result for patients. Their report appeared in the February 4 Human Molecular Genetics.

CarrotsIn both the “dry” and “wet” forms of AMD, the body is less able to temper formation of “reactive oxygen species,” molecules that fling off extra energy that damages cell parts. The enzyme superoxide dismutase 2 (SOD2), made in mitochondria in many cell types, normally supplies this antioxidant activity, but it’s deficient in some people. The National Eye Institute’s AREDS (Age-Related Eye Disease Study) recommended that patients at high risk for AMD take certain antioxidants (vitamins C and E and beta-carotene) plus zinc and copper.

The major risk factor for developing AMD, besides the “age” in the disease’s name, is smoking. Variants of three genes contribute to the risk too, but to a lesser degree. These variants combine to provide a “risk” genotype that increases chances of AMD, and a “protective” genotype. Seeing how iPS cells expressing the two genotypes differ would provide a window into the gene-environment connection, while indicating which patients can actually benefit from taking the recommended antioxidants.

The RPE, a thin layer that hugs the photoreceptors (rods and cones), is a garbage dump of sorts for broken down pigments that can generate reactive oxygen species. The rods and cones continually shed pigment-rich pieces of themselves as they break down vitamin A whilst transducing photon energy into signals to the brain.

Over time, yellowish-brown “aging” specks, collectively called lipofuscin pigments, come to pepper the cells of the RPE. These are the same dreaded “liver spots” that appear on skin as we age. One type of pigment in the RPE is called A2E. Expose it to blue light, and reactive oxygen species form.

To recapitulate AMD, the researchers created iPS cells from skin fibroblasts from four patients: 2 controls without AMD, one with two copies of the risk genotype, and a fourth participant with one protective and one risk genotype.

Speckled cells appearing in the sheet of iPS cells are RPE. (Stephen Tsang and colleagues, Columbia University)
Speckled cells appearing in the sheet of iPS cells are RPE. (Stephen Tsang and colleagues, Columbia University)

Two to three months later, RPE cells emerged from the sheets of iPS cells, appearing as a cobblestone-like pattern of pigmented cells resembling bathroom tile.

Next, to simulate aging, the researchers added A2E and blue light for 10 days to the cells, dubbed iPSC-RPE. Watching the sped-up aging showed the pathology in a way that isn’t possible probing the damaged RPEs in eyes from eye banks that have stripped off the corneas or from autopsies on long-blind patients. Those RPEs, if they are there at all, are typically shredded into uselessness.

A battery of tests chronicled the iPSC-RPE cells aging. Microscopy showed lipofuscin pigments accumulating, and mass spectrometry revealed the spectrum of proteins in the cells from the 3 types of patients – fully protective genotype and healthy, and fully at-risk and the hybrid.

Superoxide dismutase (SOD2), our antioxidant enzyme
Superoxide dismutase (SOD2), our antioxidant enzyme

The cells from the two controls poured out SOD2 after A2E exposure, explaining why those two individuals don’t have AMD. But the cells from the AMD patients made only negligible amounts of the antioxidant enzyme. And that distinction creates a biology-based, personalized approach to taking supplements. “Instead of giving AREDS cocktails, we can now do a skin biopsy and then give antioxidants only to those who have poor SOD2 responses,” Dr. Tsang told me.

1. It’s a great example of personalized medicine – with the caveat that antioxidants protect against conditions other than AMD.

2. It validates a technique that I once harbored qualms about: genome-wide association studies (GWAS).

The awkwardly-named method identifies parts of the genome that people with a particular trait or illness share, pointing to regions where causative genes may lie. Early on, GWAS results were sometimes retracted after adding data dispelled the associations. And many GWAS identified gene variants that contribute only tiny degrees to a trait. But more recently, as the numbers have grown (participants profiled and genome regions probed), GWAS have indeed led to identifying genes of interest.

The functions of the three genes considered in this study (CFH, ARMS2 and HTRA1) found using GWAS, weren’t known. But the iPSC-RPE cells clearly demonstrated their role in the antioxidant response – when mutant, cells of the RPE can’t handle the oxidative stress of accumulating aging pigments.

3. The study elegantly shows how the cell bridges the molecular and the medical. Clinical researchers can study a disease in people, geneticists can sequence the underlying faulty instructions, and molecular biologists can decipher the biochemical pathways that detour, causing disease. But watching that disease unfold in cells really reveals the pathology.

Reprogrammed, patient-derived, iPS cells provide that priceless peek – with nary an embryo in sight.


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