Japanese researchers from Gifu Pharmaceutical University and Gifu University have reported that a type of protein found in stem cells taken from adipose (fat) tissue can reverse and prevent age-related, light-induced retinal damage in mice. These results may lead to treatments for patients faced with permanent vision loss.
According to the work done by these two research teams led by Drs. Hideaki Hara and Kazuhiro Tsuruma, a single injection of fat-derived stem cells (ASCs) reduced the retinal damage induced by light exposure in mice. This study also discovered that when fat-derived stem cells were grown in culture with retinal cells, the stem cells prevented the retinal cells from suffering damage after exposure to hydrogen peroxide and visible light both in the culture and in the retinas of live mice.
Additionally, Hara and Tsuruma and their colleagues discovered a protein in fat-derived stem cells called “progranulin.” This protein, progranulin, seems to play a central role in protecting other cells from suffering light-induced eye damage.
In the retina, which lies at the back of the eye, excessive light exposure causes degeneration of the photoreceptor cells that respond to light. Several studies have suggested that a long-term history of exposure to light might be an important factor in the onset of age-related macular degeneration. Photoreceptor loss is the primary cause of blindness in particular eye-specific degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.
“However, there are few effective therapeutic strategies for these diseases,” Hideaki Hara, Ph.D., R.Ph., and Kazuhiro Tsuruma, Ph.D., R.Ph.
“Recent studies have demonstrated that bone marrow-derived stem cells protect against central nervous system degeneration with limited results. Just like the bone marrow stem cells, ASCs also self-renew and have the ability to change, or differentiate, as they grow. But since they come from fat, they can be obtained more easily under local anesthesia and in large quantities.”
The fat tissue used in the study was taken from mice and processed in the laboratory to isolate the fat-based stem cells. Afterwards, those cells were tested with cultured mouse retinal cells, and they show a robust protective effect. These successes suggested to the team to test their theory on a live group of mice that had retinal damage after exposure to intense levels of light.
Five days after receiving injections of the fat-based stem cells, the animals were tested for photoreceptor degeneration and retinal dysfunction. The results showed the degeneration had been significantly inhibited.
“Progranulin was identified as a major secreted protein of ASCs, which showed protective effects against retinal damage in culture and in animal tests using mice,” Drs. Hara and Tsuruma said. “As such, it may be a potential target for the treatment of degenerative diseases of the retina such as age-related macular degeneration and retinitis pigmentosa. The ASCs reduced photoreceptor degeneration without engraftment, which is concordant with the results of previous studies using bone marrow stem cells.”
“This study, suggesting that the protein progranulin may play a pivotal role in protecting against retinal light-induced damage, points to the potential for new therapeutic approaches to degenerative diseases of the retina,” said, Anthony Atala, MD, editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine, where this work was published.
Embryonic stem cells can form several different types of eye-specific cells. In the early years of the 21st century, reproducible and efficient methods for differentiating embryonic stem cells into lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells were developed (Haruta M., Embryonic stem cells: potential source for ocular repair. Semin Ophthalmol. 2005 Jan-Mar;20(1):17-23).
Other experiments showed that embryonic stem cells could be differentiated into neural progenitor cells (NPCs). These NPCs differentiated in culture and some of them even expressed genes characteristic of developing retinal cells. Although it must be noted that this was uncommon and cells expressing markers of mature photoreceptors were not observed. Implantation of these differentiated NPCs into the retinas of laboratory animals allowed them to survive for at least 16 weeks, migrate over large distances, and form photoreceptor-like cells that made blue-absorbing pigments. These cells also integrated into the host retina (Banin E, Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells. Steem Cells. 2006 Feb;24(2):246-57).
These early experiments were followed by several others that showed equally remarkable promise. Workers in Takahashi’s laboratory in Kobe, Japan found that embryonic stem cells could form retinal precursors, but that they rarely formed photoreceptors unless they were treated with extracts from embryonic retinas. However in a follow-up paper in 2008, Takahashi, research group found that specific cocktails of small molecules and/or growth factors could push retinal precursors to form photoreceptors (Osakada, et al., Nat Biotechnol. 2008 Feb;26(2):215-24). Kunisada’s lab in Gifu, Japan used various techniques to differentiate embryonic stem cells in culture so that they would form an elaborate retinal-like structure. When this structure was transplanted into the eyes of rodents with inherited eye diseases, these transplanted cells regenerated the ganglion cells in the retina (Aoki H, et al., Graefes Arch Clin Exp Ophthalmol. 2008 Feb;246(2):255-65). Yu’s lab from Seoul National University, Seoul, South Korea made pure RPE cell cultures from embryonic stem cells and then transplanted them into the eyes of rodents with RPE-based retinal degeneration diseases (Park UC, et al., Clin Exp Reprod Med. 2011 Dec;38(4):216-21). The transplanted cells formed RPEs and integrated into the retinas of the laboratory animals. Sophisticated functional assays definitively showed that the RPEs made from embryonic stem cells gobbled up the old segments from photoreceptors and recycled the components back to the photoreceptors (Carr AJ, et al., Mol Vis. 2009;15:283-95).
Using embryonic stem cells to make retina-like structures in culture can provide a model for testing new drugs and procedures to treat degenerative eye diseased such a macular degeneration. Also, such structures might be used to transplant sections of retina into the eyes of individuals where the retina has died off.
With this goal in mind, researchers at the University of Wisconsin-Madison have succeeded in making made early retina structures that contain growing neuroretinal progenitor cells. The novelty in this experiment is that they did it using induced pluripotent stem (iPS) cells that were derived from human blood cells.
In 2011, the laboratory, of David Gamm lab, pediatric ophthalmologist and senior author of the study whose lab is at the Waisman Center, created structures from the most primitive stage of retinal development using embryonic stem cells and iPS cells derived from human skin. These structures generated the major types of retinal cells, including photoreceptors, they did not possess the layered structure found in more mature retina. Clearly something was missing t form a retinal-like structure.
The iPS cells used in this study were made by scientists at a biotechnology company called Cellular Dynamics International (CDI) of Madison, Wisconsin. CDI pioneered the technique to convert blood cells into iPS cells, and they extracted a type of blood cell called a T-lymphocyte from donor samples. These T-lymphocytes were reprogrammed into iPS cells (full disclosure: CDI was founded by UW-Madison stem cell pioneer James Thomson).
With these iPS cells, Gamm and postdoctoral researcher and lead author Joseph Phillips, used their previously-established protocol to grow retina-like tissue from iPS cells. However, this time, about 16% of the initial retinal structures developed distinct layers, which is the structure observed in a mature retina. The outermost layer primarily contained photoreceptors, whereas the middle and inner layers harbored intermediary retinal neurons and ganglion cells, respectively. This particular arrangement of cells is reminiscent of what is found in the back of the eye.
These retinal structures also showed proper connections that could allow the cells to communicate information. In the retina, light-sensitive photoreceptor cells along the back wall of the eye produce impulses that are ultimately transmitted through the optic nerve and then to the brain, and this allows. Because these layered retinal structures not only had the proper cell types, but also the proper connections, these findings suggest that it is possible to assemble human retinal cells into the rather complex retinal tissues found in an adult retina. This is extremely stupefying when one considers that these structures all started from a single blood sample.
There are several applications to which these structures might be subjected. They could be used to test drugs and study degenerative diseases of the retina such as retinitis pigmentosa (a major cause of blindness in children and young adults). Also, it might be possible one day to replace multiple layers of the retina in order to help patients with more widespread retinal damage.
Gamm said, “We don’t know how far this technology will take us, but the fact that we are able to grow a rudimentary retina structure from a patient’s blood cells is encouraging, not only because it confirms our earlier work using human skin cells, but also because blood as a starting source is convenient to obtain. This is a solid step forward.” He also added, “We were fortunate that CDI shared an interest in our work. Combining our lab’s expertise with that of CDI was critical to the success of this study.”
This work was published in the March 12, 2012 online issue of Investigative Ophthalmology & Visual Science. The research is supported by the Foundation Fighting Blindness, the National Institutes of Health, the Retina Research Foundation, the UW Institute for Clinical and Translational Research, the UW Eye Research Institute and the E. Matilda Ziegler Foundation for the Blind, Inc.