Stem Cell-Derived Retinal Grafts Integrate into Damaged Monkey Retinas


Retinal degenerations are the leading cause of blindness and fixing a defective retina is not an easy task.

Fortunately, a model system in nonhuman primates that has been used to test retinal replacement with stem cell-derived retinal cells has seen some success. In several experiment in small animals, retinal transplantations helped blind animals regain their sight. However, small laboratory rodents are not terribly good model systems for human eye problems.

To address the clinical relevancy of this transplantation system, Shirai and colleagues confirmed in rats and in macaques that transplantion of human embryonic stem cell (hESC)–derived retinas integrate into the already-existing retina and develop as fully mature retinal grafts.

In this paper, Shirai and others established the developmental stage at which embryonic stem cell-derived retinal cells could integrate into the retina and replace damaged cells. By transplanting cells into nude rats that do not have the ability to reject transplanted tissue, they refined their cell-based technique to heal damaged retinas. Then they took their refined technique into macques to treat two newly established monkey models of retinal degeneration.

In the first model system, Shirai et al. exposed one group monkeys to retina-damaging chemicals, and the other group had their retinas damaged by lasers. In both cases, the result was photoreceptor degeneration. Anywhere from 46 to 109 days after injury, the human embryonic stem cell-derived retinal sheets were implanted into the damaged retinas.

The retinal grafts integrated into the primate eyes and continued to differentiate into cone and rod cells, which are the two types of photoreceptor cells in the retina. Functional studies are still being conducted, but if vision can be improved, but these new macaque models confirm the clinical potential of stem cell–derived grafts for retinal blindness that results from photoreceptor degeneration.

See H. Shirai et al., Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration. Proc. Natl. Acad. Sci. U.S.A. 113, E81–E90 (2015).

Embryonic Stem Cell-Derived Retinal Cells Treat Blindness in Eye Patients


Embryonic stem cells are derived from human embryos, can only grow in culture indefinitely, and have the ability to potentially differentiate into any adult cell type in the human body.  Because cell and tissues made from embryonic stem cells bear the same tissue types as the embryos from which they were derived, they will be rejected by the immune system patient.  However, there are sites in our bodies were the immune system does not go, and that includes the central nervous system and the eyes.  This is the reason why clinical trials with embryonic stem cell-derived cells have focused, to date, on spinal cord injuries and eye diseases.

Several clinical trials have examined the ability of retinal pigmented epithelial (RPE) cells made from embryonic stem cells to treat patients with dry macular degeneration or an inherited eye disease called Stargardt’s disease.  Data from these trials has been reported in an article in the medical journal The Lancet, and accordingly, none of the treated patients showed tumor formation or immunological rejection of the implants and, most impressively perhaps, partial blindness was reversed in about half of the eyes that received transplants.

The results might re-energize the quest to harness embryonic stem cells for human medicine.  Dr. Anthony Atala of the Wake Forest Institute for Regenerative Medicine called the work “a major accomplishment” in an accompanying commentary on the article.

RPE cells lie just behind the photoreceptor cells in the retina of our eyes.  Photoreceptors have their ends hurried in the RPE layer.  This arrangement exists for a very good reason; the photoreceptors are exposed to high intensities of light and they suffer respectable amounts of oxidative damage.  The components of the photoreceptors cells are made in the very lowest parts of the RPEs and then are eventually pushed to the ends of the cells.  At the end of the photoreceptor cells, the RPEs relieve the photoreceptors of their photodamaged parts and gobble them down, and recycle the cellular components.  Thus, RPE cells serve a photoreceptor cell repair and service cells.  If the RPE cells begin to die, the photoreceptors are not long the this work either.

In the case of dry macular degeneration, which accounts for 90 percent of diagnosed cases of macular degeneration, the light-sensitive photoreceptor cells of the macula (the portion of the retina were the day vision is the sharpest) slowly break down. Damage to the macula causes blurring or spotty loss of central vision and yellowish cellular deposits called drusen (extracellular waste products from metabolism) form under the retina between the retinal pigmented epithelium (RPE) layer and a basement membrane called Bruch’s membrane, that supports the retina. An increase in the size and number of drusen is associated with the death of RPE and, consequently, photoreceptor cells, and is sometimes the first sign of dry macular degeneration.

Medical illustration of dry macular degeneration

Mutations in several genes have been identified in families with dry macular degeneration that increase the risk for dry macular degeneration.  These include the SERPING1 gene, those genes that encode the complement system proteins  factor H (CFH), factor B (CFB) and factor 3, and fibulin-5.  Additionally, some environmental and behavioral factors also influence the risk a person will develop macular degeneration.  These include smoking, exposure to blue light, ingestion of a high-fat diet, elevated blood pressure and serum cholesterol levels, and low vitamin D levels.

Stargardt’s disease is an inherited, juvenile form of macular degeneration that is caused by mutations in the ABCR gene.  The protein encoded by this gene is a waste metabolite transporter, and defects in this protein cause the build up of a toxic metabolite called lipofuscin in the RPE cells, which leads to their demise and the death of the photoreceptors.

In this study, the main goal was to assess the safety of the transplanted cells. The study “provides the first evidence, in humans with any disease, of the long-term safety and possible biologic activity” of cells derived from embryos, said co-author Dr. Robert Lanza, chief scientific officer of Advanced Cell Technology, which produced the cells and funded the study.

Nine patients with Stargardt’s disease and nine with dry age-related macular degeneration received implants of the retinal cells in one eye. The other eye served as a control.  Four eyes developed cataracts and two became inflamed, probably due to the patients’ age (median: 77) or the use of immune-supressing transplant drugs.

The implanted RPE cells survived in all 18 patients, most of whose vision improved.  In those with macular degeneration, treated eyes saw a median of 14 additional letters on a standard eye chart a year after receiving the cells, with one patient gaining 19 letters. The untreated eyes got worse, overall. The Stargardt’s patients had similar results.

In real-life terms, patients who couldn’t see objects under 12 feet (4 meters) tall can now see normal-size adults.

The vision of one 75-year old rancher who was blind in the treated eye (20/400) improved to 20/40, enough to ride horses again, Lanza said.  Others became able to use computers, read watches, go to the mall or travel to the airport alone for the first time in years.

While calling the results “encouraging,” stem cell expert Dusko Ilic of Kings College London, who was not involved in the work, warned that even if the larger clinical trial planned for later this year is also successful, “it will take years before the treatment becomes available.”

Other cell types can also form RPE cells and these include induced pluripotent stem cells, mesenchymal stem cells from fat (Ophthalmic Res. 2012;48 Suppl 1:1-5), adult retinal stem cells (Pigment Cell Melanoma Res. 2011 Feb;24(1):233-40), and iris pigmented epithelial cells (Prog Retin Eye Res. 2007 May;26(3):302-21).  We do not need to destroy embryos to treat eye diseases with stem cells.

An Easy Way to Make Retinal Pigment Epithelium from Pluripotent Stem Cells


Age-related macular degeneration is the leading cause of irreversible vision loss and blindness among the aged in industrialized countries. One of the earliest events associated with age-related macular degeneration (AMD) is damage to the retinal pigmented epithelium (RPE), which lies just behind the photoreceptor cells in the retinal. The RPE serves several roles in visual function, including absorption of stray light, formation of blood retina barrier, transport of nutrients, secretion of growth factors, isomerization of retinol, and daily clearance of shed outer photoreceptor outer segments. RPE cell death and dysfunction is associated with both wet (neovascular) and dry (atrophic) forms of AMD.

How then do we make RPE cells from stem cells in order to treat AMD? In previous experiments, scientists have used RPEs made from human embryonic stem cells to treat two patients with inherited eye diseases. The results from these experiments were underwhelming to say the least. Also, the derivation of RPEs from embryonic stem cells was tedious and laborious. Is there a better way?

Make that a yes. A paper in Stem Cells Translational Medicine from Donald Zack’s laboratory at Johns Hopkins University School of Medicine describes a simple and highly scalable process for deriving RPEs from human pluripotent stem cells.

To begin with, the cells were plated at relatively high densities (20,000 cells / cm square centimeter) in a medium called TeSR1. This medium can support the growth of human pluripotent stem cells and can also keep them undifferentiated without the use of animal feeder cell lines. SInce there are no feeder cells to make, the cultivation of these cells is much simpler than before and the variability from culture to culture decreases.

After five days of growth, the cells grew to a monolayer (the cells had grown and spread throughout the culture dish) and were transferred to a 5% carbon dioxide and 20% oxygen incubator. Three days later, they were transferred to Delbecco’s Modified Eagle Medium with F12 supplement or DMEM/F12. This culture medium supports stem cell differentiation. The cells grew and differentiated, for about 25 days, but RPEs were easily visible because they make loads of dark pigment. Once the dark colonies appeared, the cells were allowed to grow another 25 days. The cells were transferred into Delbecco’s Medium with enzymes to pull the cells apart from each other for four hours, then, after pipetting them vigorously, the cells were centrifuged, and suspended in a cell detachment solution called Accumax.

The separated cells were filtered and plated on specially coated plates, and cultured in “RPE medium.” This is a mixture of several different culture media that favors the survival and growth of RPEs. Because RPE colonies were easily seen with their dark pigments, they were specifically picked and passaged. The result was extremely clean RPE cultures from pluripotent stem cells.

Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.
Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.

The cells were subjected to a battery of tests: flow cytometry, Western blotting, Immunostaining and so on. These cells passed with flying colors and they are clearly RPE cells that express RPE-specific genes, have RPE-specific proteins on their cell surfaces, and even snuggle up to photoreceptors and recycle their terminal segments.  The final functional test came from a transplantation experiment in which human RPEs made from human pluripotent stem cells were transplanted behind the retinas of mice with impaired immune systems.  The cells, as you can see in the figures below, integrated beautifully, and were also highly functional, as indicated by the rhodopsin-positive vesicles in the implanted RPE cells.   No tumors were seen in any of the laboratory animals implanted with the stem cell-derived RPEs.

Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.
Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.

This new procedure is able to make RPEs from pluripotent stem cells in a simple and highly scalable way.  If human induced pluripotent stem cells could be used with this protocol, and there seems little reason that should not be highly possible, then such cells could be easily used for human clinical trials.