When pluripotent stem cells are differentiated into photoreceptor cells, and then implanted into the retina at the back of the eye of a laboratory animal, they do not always survive. However, pre-treatment of those cells with an antiaging glycoprotein (AAGP), made by ProtoKinetix, causes those transplanted cells to be 300 times more viable than cells not treated with this protein according to a study recently accepted for publication.
AAGP was invented by Dr. Geraldine-Castelot-Deliencourt and developed in partnership with the Institute for Scientific Application (INSA) of France. For her work in this area Dr. Castelot-Deliencourt was honored with France’s highest award for scientific accomplishment, the Francinov Award, in 2006.
AAGP significantly improves the viable yield of stem cells transplanted in retinal tissue, according to experiments conducted at the University of British Columbia in the laboratory of Dr. Kevin Gregory-Evans.
AAGP seems to protect cells from inflammation-induced cell death. This is based on experiments in which cultured cells that were treated with AAGP were significantly more resistant to hydrogen peroxide, ultraviolet A (wavelengths of 320-400 nanometers), and ultraviolet C (shorter than 290 nm). In addition, when exposed to an inflammatory mediator, interleukin β (ILβ), AAGP exposure reduced COX-2 expression three-fold. COX-2 is an enzyme that is induced by the various stimuli that stimulate Inflammation. It is, therefore, an excellent read-out of the degree to which inflammation has been induced. The fact that AAGP prevented the induction of COX-2 shows that this protein can inhibit the induction of inflammation. These data suggest that AAGP™ may not just be usable in cell and organ storage but also in pharmacological treatments.
A presentation at the annual meeting of the Association for Research in Vision and Ophthalmology in Seattle, Washington has reported the safe transplantation of stem cells derived from a patient’s skin to the back of the eye in an effort to restore vision. The subject for this research project suffered from advanced wet age-related macular degeneration that did not respond to current standard treatments.
A small skin biopsy from the patient’s arm was collected and reprogrammed into induced pluripotent stem cells (iPSCs). The iPSCs were then differentiated into retinal pigmented epithelial (RPE) cells, which were transplanted into the patient’s eye. The transplanted cells survived without any adverse events for over a year and resulted in slightly, though significantly, improved vision.
iPSCs are adult cells that have been reprogrammed to an embryonic stem cell-like state, which can then be differentiated into any cell type found in the body.
Abstract Title: #3769: Transplantation of Autologous induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Sheets for Exudative Age Related Macular Degeneration: A Pilot Clinical Study by Yasuo Kurimoto and others from the laboratory of Masayo Takahashi’s laboratory at the RIKEN Center for Developmental Biology in Kobe, Japan.
Unfortunately, this clinical trial has been suspended because iPSCs made from other patients proved to possess too many genetic abnormalities. Therefore, Takahashi and her colleagues have decided that allogeneic iPSCs differentiated into RPEs will probably do a better job than the patient’s own skin cell-derived iPSCs.
Collaboration between scientists from mainland China, the University of California, San Diego School of Medicine and Shiley Eye Institute have developed a new, stem cell-based technique that permits remaining stem cells to regrow functional lenses after the diseased lens was removed. This treatment was initially tested in laboratory animals, but it has now been tested in a small human clinical trial. This procedure produced far fewer surgical complications than the current standard-of-care. The real boost is that this regenerative procedure resulted in regenerated lenses that had superior visual qualities in all 12 of the pediatric cataract patients who served as subjects for this clinical trial.
Kang Zhang, MD, PhD, chief of Ophthalmic Genetics, founding director of the Institute for Genomic Medicine and co-director of Biomaterials and Tissue Engineering at the Institute of Engineering in Medicine, both at UC San Diego School of Medicine, said: “An ultimate goal of stem cell research is to turn on the regenerative potential of one’s own stem cells for tissue and organ repair and disease therapy.” Zhang and his colleagues published their work in the journal Nature.
Cataracts are cloudiness over the lens of the eye that blurs vision. The lens consists mostly of water and protein. When the protein aggregates, it clouds the lens and reduces the light that reaches the retina. This clouding may become severe enough to cause blurred vision. Most age-related cataracts develop from protein clumpings. You do not have to be older to suffer from cataracts. Congenital cataracts occur at birth or shortly after birth. Scarring of the retina or prenatal damage to the eye can cause congenital cataracts. Congenital cataracts are a significant cause of blindness in children. Current treatment for congenital cataracts is limited by the age of the patient. Most pediatric patients require corrective eyewear after cataract surgery.
To address this medical need, Zhang and colleagues examined the regenerative potential of endogenous stem cells on the lens. Unlike other stem cell approaches that involve creating stem cells in the lab and introducing them back into the patient, Zhang decided to use stem cells that are already in place at the site of the injury to do the heavy lifting. In the human eye, lens epithelial stem cells or LECs generate replacement lens cells throughout a person’s life, even though their production declines with age.
Unfortunately, current cataract surgeries essentially remove LECs within the lens. Whatever cells might be left over produce disorganized regrowth in infants and no useful vision. Zhang and his colleagues first confirmed that LECs had regenerative potential. To confirm this, they used laboratory animals. With that knowledge in hand, Zhang and his collaborators devised a novel, minimally invasive surgical procedure that removes the cloudy lens, but manages to maintain the integrity of the membrane that gives the lens its required shape (the lens capsule). With the lens capsule in place, the LECs were activated to replace the missing lens.
Once again, Zhang and his team ensured that their technique worked in animals before they ever tried it on a human patient. Animals with cataracts whose lenses were extirpated, but whose lens capsules were left intact, regenerated new lenses that were devoid of cataracts and provided excellent sight. With their technique honed and ready, Zhang and others tested their procedure on very young human infants in a small human trial. They discovered that their new surgical technique allowed pre-existing LECs to efficiently regenerate functional lenses. In particular, the human trial involved 12 infants under the age of 2 treated with the new method developed by Zhang and others, and 25 similar infants receiving current standard surgical care.
The results were stark: the control group experienced a higher incidence of post-surgery inflammation, early-onset ocular hypertension and increased lens clouding, but those infants who received Zhang’s new procedure showed fewer complications and faster healing. After three months, the 12 infants who underwent the new procedure had a clear, regenerated biconvex lens in all of their eyes.
“The success of this work represents a new approach in how new human tissue or organ can be regenerated and human disease can be treated, and may have a broad impact on regenerative therapies by harnessing the regenerative power of our own body,” said Zhang.
Zhang indicated that he and his colleagues are now looking to apply what they learned in this project to tackling the issue of age-related cataracts. Age-related cataracts are the leading cause of blindness in the world. Over 20 million Americans suffer from cataracts, and more than 4 million surgeries are performed annually to replace the clouded lens with an artificial plastic lens (intraocular lens).
Despite technical advances, a large portion of patients undergoing surgery are left with suboptimal vision post-surgery and are dependent upon corrective eyewear for driving a car and/or reading a book. “We believe that our new approach will result in a paradigm shift in cataract surgery and may offer patients a safer and better treatment option in the future,” said an optimistic Zhang.
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).
Masayo Takahashi, an ophthalmologist at the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, has pioneered the use of induced pluripotent stem cells (iPSCs) to treat patients with degenerative retinal diseases.
Takahashi isolated skin cells from her patients, and then had them reprogrammed into iPSCs in the laboratory through a combination of genetic engineering and cell culture techniques. These iPSCs have many similarities with embryonic stem cells, including pluripotency, which is the potential to differentiate into any adult cell type.
Once induced pluripotent stem cell lines were established from her patient’s skin cells, they had their genomes sequenced for safety purposes, and then differentiated into retinal pigmented epithelial (RPE) cells. RPE cells lie beneath the neural retina and support the photoreceptors that respond to light. When the RPE cells die off, the photoreceptors also begin to die.
Takahashi watched the transplantation of the RPE cells that she had grown in the laboratory into the back of a woman’s damaged retina. This transplant would constitute the first test of the therapeutic potential of iPSCs in people. Takahashi described the transplant as “like a sacred hour.”
Takahashi has collaborated with Shinya Yamanaka, the discoverer of iPSC technology. She devised ways to convert the iPS cells into sheets of RPE cells. She then tested the resulting cells in mice and monkeys, jumped the various regulatory loops, recruited patients for her clinical trial, and practiced growing cells from those patients. Finally, she was ready to try the transplants in people with a common condition called age-related macular degeneration, in which wayward blood vessels destroy photoreceptors and vision. The transplants are meant to cover the retina, patch up the epithelial layer and support the remaining photoreceptors. Watching the procedure, “I could feel the tension of the surgeon,” Takahashi said.
This transplant surgery occurred approximately a year ago. Some new data on this patient is available.
As of 6 months after the transplant, the procedure appears to be safe. The one-year safety report should appear soon. Prior to the transplant, the patient was a series of 18 anti-vascular endothelial growth factor (anti-VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease. However, data presented by Dr. Takahashi showed that the patient had subretinal fibrotic tissue removed during the transplant surgery in order to make room for the RPE cells. Once the RPE cells were implanted, the patient experienced no recurrence of neovascularization at the 6-month point. This is significant because she has not had any other anti-VEGF injections since the transplant. Her visual acuity was stabilized and there have been no safety related concerns to date.
I must grant that this is only one patient, but so far, these results look, at least hopeful. Hopefully other patients will be treated in this trial, and hopefully, they will experience the same success that the first patient is enjoying. We also hope and pray that the first patient will continue to experience relief from her retinal degeneration.
As to the treatment of the second patient of this trial, Takahashi has hit a snag. Some mutations were detected in the iPS cell-derived RPE cells prepared for the second patient. No one knows if these mutations make these cells dangerous to implant. Regulatory guidelines, at this point, are also no help. Apparently, the cells have three single-nucleotide change and three copy-number changes that are present in the RPE cells that were not detectable in the patient’s original skin fibroblasts. The copy-number changes were, in all cases, single-gene deletions. One of the single-nucleotide changes is listed in a database of cancer somatic mutations, but only linked to a single cancer. Further evaluation of these mutations shows that they were not in “driver genes for tumor formation,” according to Dr. Takahashi.
Tumorigenicity tests in laboratory animals has established that the RPE cells are safe. Remember that the presence of a mutation does not necessarily mean that these RPE cells can be tumorigenic.
However, Takahashi has still decided to not transplant these cells into the second patients. Part of the reason is caution, but the other reason is compliance with new Japanese law on Regenerative Medicine, which became effective after iPS trial was begun. This law, however, does not specify how safe a cell line has to be before it can be transplanted into a patient.
RIKEN’s decision to halt the trial is probably a good idea. After all, this is the first trial with iPSCs and it is important to get it right. Even though the RPE cells were widely thought to be safe to use, Takahashi decided not to implant another patient with RPEs derived from their own cells. Instead, they decided to use RPEs made from donated iPSC lines. Therefore, Takahashi is in discussions government officials to determine how this change of focus for the trial affects their compliance with Japanese law.
Frankly, this might be a very savvy move on Takahashi’s part. As Peter Karagiannis, a spokesperson for the Center for iPS Cell Research and Application, noted: “As of now, autologous would not be a feasible way of providing wide-level clinical therapy. At the experimental level it’s fine, but if it’s going to be mass-produced or industrialized, it has to be allogeneic.”
Therefore, the RIKEN institute is moving forward with allogeneic iPSC-derived RPEs. RIKEN will work in collaboration with the Center for iPS Cell Research and Application (CiRA) in Kyoto, Japan, which has several well characterized, partially-matched lines whose safety profiles have been established by strict, rigorous safety testing methods. However, immunological rejection remains a concern, even if these cells are transplanted into an isolated tissue like the eye where to immune system typically is not allowed. The simple fact is that no one knows if the cells will be rejected until they are used in the trial.
An additional concern is that CiRA has not typed its cells for minor histocompatibility antigens, which can cause T cell–mediated transplant rejection.
Nevertheless, Takahashi and her team deserve a good deal of credit for their work and vigilance.
If we take tissue samples from the mouth and grow them in the laboratory and manipulate them, we might be able to cure the blind. Blind people who suffer from stem cell deficiency in the cornea might be able to see again by using stem cells isolated from the mouth. Furthermore, this treatment might not only restore vision, but it might also ameliorate pain in the cornea.
Ophthalmologist Tor Paaske Utheim has conducted research for over ten years on how to cure certain types of blindness by using stem cells harvested from tissue obtained from different parts of the body. He then transplants this cultured tissue into the damaged eye, and patients who suffer from blindness as a result of corneal stem cell deficiencies can regain their sight. Recently, Utheim’s research has utilized stem cells from the mouth to grow new corneal tissue, and has also tried to design optimal methods to store and transport this tissue to treat patients.
Utheim is the head of a research group at the Faculty of Dentistry at the University of Oslo (UiO) and the Department of Medical Biochemistry at Oslo University Hospital.
Using cells extracted from the mucous membrane lining the inside of the mouth (the oral mucosa) can restore vision is new to most people. Only ten years ago, this was considered impossible, but results confirm the potential of this method. Twenty clinical studies from various countries have, to date, shown good results, according to Utheim. These clinical trials, however, have only applied these cells to a group of diseases caused by stem cell deficiency in the cornea.
Utheim and his colleagues hope to treat patients with eye injuries caused by so-called limbal stem cell deficiencies. This disorder can be caused by such things as UV radiation, chemical burns, serious infections like trachoma, and various other diseases, some of which are heritable. The number of people worldwide affected by limbal stem cell deficiency is unknown, but in India alone there is an estimated 1.5 million. This disorder most often affects people living in developing countries.
Stem cells that are found at the outer edge of the cornea help to keep the surface of the cornea even and clear. In limbal stem cell deficiencies, the stem cells have been damaged, and they cannot renew the cornea’s outermost layer. Instead, other cells grow over the cornea, which clouds the cornea. The cornea can become fully or partially covered, explains Utheim, which leads to impaired vision or blindness.
Others suffer from severe pain as well. When one patient was interviewed by Norwegian national broadcaster NRK about his limbal stem cell deficiency, he responded: “I don’t know what’s worse: the pain, or losing my sight.”
Utheim explained that when stem cells do not work properly, ulcers can develop in the cornea, which exposed nerve fibers. Since the number of nerve fibers is far higher in the cornea than for example in the skin, it is not surprising that some patients experience severe pain.
A breakthrough within the field occurred about ten years ago when Japanese researchers showed that cells from the oral mucosa could be used to replace limbal stem cells in patients with limbal stem cell deficiency. Although it had been possible since the late 1990s to cure the disorder using cultured stem cells. The available treatment relied on the patient having a healthy eye from which to collect cells.
Further developments made it possible to harvest cells from a relative or deceased individual, but using limbal stem cells from other patients required the use of strong immunosuppressive drugs for the patients, which could cause serious side effects.
A milestone seemed to be reached when it became possible to use a patient’s own cells to treat blindness in both eyes without the need for immunosuppressive drugs. Strangely, this makes some sense because there are similarities between the oral mucosae and the surface of the eye (see Utheim TP. Stem Cells. 2015;33:1685-1695). Originally, using mouth mucosal cells to treat the eye required that the laboratory where the cells are cultured and the clinic where the patients are treated be quite close together. Because there were no protocols for storing extracted oral mucosal cells so that they can be easily kept and transported. This has made the treatment virtually inaccessible to many of the patients who need it the most, namely those in developing countries. However, this may be about to change.
Utheim’s research group is now on the brink of a development that will make it possible to cure both severe pain and blindness in patients who are spread over a larger geographical area than before (see Islam R, et al. PLoS One. 2015;10:e0128306.). “Today, cells from the mouth are cultured for use in the treatment of blindness in only a few specialized centers in the world. By identifying the optimal conditions for storing and transporting the cultured tissue, we would allow for the treatment to be made available worldwide, and not just close to the cell culture centers,” said Rakibul Islam, who is a PhD candidate in the Department of Oral Biology at the Faculty of Dentistry.
Islam is collaborating with Harvard Medical School to introduce this method of treating blindness to clinics around the world. Islam’s findings could also help improve treatment outcomes. “Being able to store the cultured tissue in a small sealed container for a week increases the technique’s flexibility significantly. It makes it easier to plan the operation and allows for quality assurance through microbiological testing of the tissue before transplantation,” Islam explained.
One of the things that Islam and his colleagues have discovered is the specific temperature range at which cells from the mouth should ideally be stored at after culture. Islam has shown that cultured mouth stem cells retain their quintessential properties best between 12 and 16 degrees Celsius (See Dolgin, Elie. Nature Biotechnolgy, 2015;33:224-225.).
During a brief stint at Harvard University, Islam also examined which areas of the mouth are best suited to use in regenerative medicine. In other words, Islam and his colleagues wanted to know which parts of the mouth contain cell layers that regenerate the fastest. Islam explained this using this example: “If you burn any part of your mouth on hot coffee, it heals so quickly that by the next morning you have forgotten about it. This is because the oral mucosa contains cells that multiply quickly. We wanted to investigate whether there were regional differences in the mouth that we could exploit for the treatment of limbal stem cell deficiency.”
Islam continued, “Our results show that the location from which the mucosal tissue is harvested has a striking impact on the quality of the cultured tissue.”
The results from this particular study have not yet been published.
This research can potentially give hope to the many blind that live far away from centralized cell culture laboratories. In work by Utheim in 2010, in collaboration with the ophthalmologist Sten Ræder, he developed storage technology for cultured stem cells that enables the cultured tissue to be transported in a small custom-made plastic container. Tissue from stem cells is thus freed from expensive and bulky laboratory equipment and provides a whole new level of flexibility.
Utheim said “The sample of cells from the mouth can be sent by air over long distances to specialist laboratories with first-class equipment and expertise. After a couple of weeks of laboratory cultivation, the sender may receive the tissue back ready for use. An ophthalmologist could then transplant the stem cells onto the patient’s eye.”
However, the container was just one step in the right direction: “Now we have identified those areas of the mouth that may be best suited for regenerative medicine, and developed a method for storing and transporting tissue from centralized, highly specialized tissue culture centers to clinics worldwide. Our findings are helping to simplify and streamline the clinical procedures, and to make the treatment far more accessible than it is today,” said Islam, who admitted that the transport potential of the project has been integral to his own enthusiasm. He continued, “Although the scientific and technical aspects of our project are very exciting, it has been especially motivating to think of the possibilities this storage technology brings to treating blindness in all parts of the world, including my homeland Bangladesh.”
A central laboratory for the growth of stem cells already exists in Italy. In fact, earlier this year the European Medicines Agency approved the procedure for the cultivation of stem cells from the cornea in EU laboratories. This is the first stem cell therapy to be approved by the European Medicines Agency, according to the journal Nature Biotechnology. Utheim described the approval as an important step towards the implementation of stem cell technology over larger geographical areas. To date, almost 250 people with limbal stem cell deficiency have undergone treatment involving transplantation of stem cells grown from their own mouth cells. “This provides a good basis for judging the success of the treatment” Utheim says.
He has recently published an article in the journal Stem Cells on the inherent potential of cells from the mouth to regenerative medicine. Roughly three out of four treatments are described as successful.
Stem cell transplantation is a promising potential treatment for retinal degenerative diseases. Because retinal degeneration often leads to blindness, stem cells might be one of the up-and-coming tools in the battle against blindness.
The laboratory of Shaomei Wang (Cedars-Sinai Medical Center, Los Angeles) have assessed the effectiveness of stem cell-based therapeutic strategies using the Royal College of Surgeons (RCS) rat model, which mimics the disease progression of age-related macular degeneration (AMD). In RCS mice, the retinal pigment epithelium or RPE degenerates and is disrupted, which leads to the death of photoreceptors (Mullen RJ and LaVail MM Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 1976;192:799-801). The work by Wang and his colleagues has shown that human cortical-derived neural progenitor cells (hNPCctx) could dramatically rescue vision in the RCS rat (see Wang S, and others, Investigative ophthalmology & visual science 2008;49:3201-3206; Gamm DM, and others, Wang S, Lu B, et al. PLoS One 2007;2:e338). Unfortunately, the fetal origin of these cells presents an obstacle, because such cells are not readily available and come from aborted fetuses.
To overcome such obstacles, Wang and his colleagues assessed the ability of a stable neural progenitor cell line (iNPCs) derived from induced pluripotent stem cells (iPSCs) to preserve vision after sub-retinal injection into RCS rats (Sareen D, and others, J Comp Neurol 2014;522:2707-2728). A report in the journal Stem Cells by Wang and others establishes that iNPC injection leads to the reversal of AMD-related symptoms, the preservation of visual function, and may represent a patient-specific therapeutic option (Tsai Y, and others, Stem Cells 2015;33:2537-2549).
Wang and his others showed that an iNSC-treated eye scored higher in all functional tests used (optokinetic response (OKR), electroretinography (ERG), and luminance threshold responses (LTR)), compared to an untreated eye, in RCS mice at 150 days post-transplant. This improvement nicely correlates with the improved protection of photoreceptors in iNPC-treated eyes, which presented with normal cone morphology and the reversal of disease-associated changes throughout the retina.
So how do iNPCs help preserve the photoreceptors and visual function? Wang and his team found that iNPCs survived up to 130 days in RCS retinas, which when normalized to lifespan, represents around 16 years in humans. Additionally, they discovered that iNPCs were able to migrate to an area between the retinal pigment epithelial and photoreceptor layers. This allows the injection of cells into non-affected neighboring regions of the retina, which will not to worsen any compromised retinal components. iNPCs did, however, continue to express NSC/NPC markers and did not mature neural/retinal markers, suggesting that grafted-iNPCs remained phenotypically uncommitted progenitor cells and did not differentiate towards a retinal phenotype.
Further investigations found that iNPC-treatment reduced levels of toxic undigested bits of the photoreceptor cell membranes. Accumulation of these photoreceptor outer segments (POS) cause the photoreceptors to die off. Typically, the RPE cells goggle up these toxic membrane bits, degrade them, and recycle their components for the photoreceptors. The fact that these POS bits were not accumulating in the retinas of RCS mice suggested to Wang and his colleagues that the grafted-iNPCs restored POS degradation in RCS rats. They subsequently found that iNPCs expressed phagocytosis-related genes and could gobble up and degrade POS in culture. They extended these findings in living creatures by identifying the different stages of POS digestion and even viewed engulfed membranous discs inside the cytoplasm of iNPCs.
Overall, iNPC injection appears to be a safe and effective long-term treatment for Acute Macular Degeneration in the RCS rat preclinical model, and holds great promise for the translation into a patient-specific treatment for the preservation of existing retinal structure and vision during the early stages of AMD in humans. Wang noted that iNPC treatment in this model occurred at later stages of degeneration, which represents a more clinical relevant stage. However, an unstudied possibility is restoring phagocytosis by iNPCs to treat loss of visual acuity early on in the course of the disease.