Mouse Study Suggests Stem Cells Can Ward Off Glaucoma


Regulating the internal pressure of the eyeball (known as the “intraocular pressure” or IOP) is crucial for the health of the eye.  Failure to maintain a healthy IOP can lead to vision loss in glaucoma.  However, a new set of experiments by Dr. Markus Kuehn and his colleagues at the Iowa City Veterans Affairs Medical Center and the University of Iowa has shown that infusions of stem cells could help restore proper drainage for plugged-up eyes that are at risk for glaucoma.

Kuehn and his coworkers injected stem cells into the eyes of laboratory mice suffering with glaucoma.  These infused cells regenerated the tiny, fragile patch of tissue known as the trabecular meshwork, which functions as a drain for the eyes.  When fluid accumulates in the eye, the increase in IOP can lead to glaucoma.  Glaucoma damages the optic nerve leads to blindness.

“We believe that replacement of damaged or lost trabecular meshwork cells with healthy cells can lead to functional restoration following transplantation into glaucoma eyes,” Kuehn wrote on his lab’s website.  One potential advantage of the approach is that induced pluripotent stem cells (iPSCs) could be created from cells harvested from a patient’s own skin. That gets around the ethical problems with using fetal stem cells.  It also lessens the chance of the patient’s body rejecting the transplanted cells.

In order to differentiate iPSCs into trabecular meshwork (TM) cells, Kuehn’s team cultured the iPSCs in medium that had previously been “conditioned” by actual human trabecular meshwork cells.  Injection of these TM cells into the eyes of laboratory rodents led to a proliferation of new endogenous cells within the trabecular meshwork.  The injected stem cells not only survived in the eyes of the animals, but also induced the eye into producing more of its own TM cells, thus multiplying the therapeutic effect.

Glaucoma has robbed some 120,000 Americans of their sight, according to data provided by the Glaucoma Research Foundation.  African-Americans are at especially high risk, as are people over age 60, those with diabetes, and those with a family history of the disease.  Glaucoma can be treated with medicines, but is not curable.  Management of the disease can delay or even prevent the eventual loss of vision. Among the treatments used are eye drops and laser or traditional surgery.

Kuehn and his team think that their findings show some promise for the most common form of glaucoma, known as primary open angle glaucoma.  It remains unclear if this mouse model is as relevant for other forms of the disease.  Another possible limitation of this research is that the new trabecular meshwork cells generated from the stem cell infusion eventually succumb to the same disease process that caused the breakdown in the first place.  This would require re-treatment and it is unclear whether an approach requiring multiple treatments over time would be viable. Kuehn and others to continue investigate this potentially fruitful approach.

This paper was published in the journal Proceedings of the National Academy of Sciences:  Wei Zhu et al., “Transplantation of iPSC-derived TM cells rescues glaucoma phenotypes in vivo,” Proceedings of the National Academy of Sciences, 2016; 113 (25): E3492 DOI: 10.1073/pnas.1604153113.

Researchers Grow Retinal Ganglion Cells in the Laboratory


Researchers from laboratory of Donald Zack at The Johns Hopkins University in Baltimore, Maryland have used genome editing methods to efficiently differentiate human pluripotent stem cells into retinal ganglion cells. Retinal ganglion cells are found in the retina that and helps transmit visual signals from the eye to the brain. Abnormalities or death of ganglion cells can cause vision loss, and conditions such as glaucoma and multiple sclerosis can wreak havoc on ganglion cells.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” said Zack, who is the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

Published in the journal Scientific Reports, Zack and his team genetically modified a line of human embryonic stem cells so that they would fluoresce once they differentiated into retinal ganglion cells. Then they used these cells to develop new differentiation methods and characterize the resulting cells.

To genetically modify their cells, Zack and others used the CRISPR-Cas9 system. CRISPR stands for “clustered regularly interspaced short palindromic repeats” and these are short segments DNA, which are found in bacteria, contain short repeated sequences. Following each repeated sequence is a short spacer that usually comes from previous exposures to a bacterial virus or plasmid. Bacteria use the CRISPR/Cas system as a kind of immune system that prevents cells from being invaded by foreign DNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes.

When bacteria are invaded by a virus, the particular Cas nucleases capture the viral DNA, cut it and insert it into the CRISPR array. When the bacterial cell is infected by a virus, an RNA is transcribed from the CRISPR array called the crRNA. This crRNA then hybridizes with the invading DNA or RNA and the double-stranded RNA or DNA/RNA hybrid is degraded by Cas proteins.

The CRISPR/Cas system is a useful laboratory tool for gene editing or adding, disrupting or changing the sequences of particular genes. If Cas9 and the appropriate crRNA are delivered into cells, you can cut a genome almost anywhere. CRISPR has a huge number of potential applications.

Zack and his group used the CRISPR/Cas system to insert a fluorescent protein gene into the DNA of their stem cells line. This red fluorescent protein would be expressed if a gene called BRN3B (POU4F2) was also expressed. BRN3B is expressed by mature retinal ganglion cells. Therefore, once these cells differentiated into retinal ganglion cells, they would glow red when viewed with a fluorescence microscope.

After differentiating their cells, Zack and his coworkers used a technique called fluorescence-activated cell sorting to isolate fully differentiated cells from other cells. The pure cell culture contained cells that displayed the biological and physical properties observed in retinal ganglion cells produced naturally, according to Zack.

As an added bonus, Valentin Sluch, a former graduate student in Zack’s laboratory, and her colleagues discovered that soaking the pluripotent stem cells in a chemical called “forskolin” at the commencement of the differentiation protocol significantly improved the efficiency of differentiation. Forskolin is a labdane diterpene found in the roots of the Indian Coleus plant (Coleus forskohlii), which belongs to the mint family.  It is used by some people as a weight loss supplement by some people.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” said Sluch, who is now a postdoctoral scholar working at Novartis. Sluch continued, “I was very excited when it first worked. I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for Multiple Sclerosis, Zack is collaborating with Dr. Peter Calabresi, professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

Stem Cell Clinical Trials in 2014


Dr. Alexey Bersenev has done the stem cell community a great service by compiling the clinical trials that involved the used of stem cells for 2011-2014.

In 2014, there were 373 clinical trials registered in international databases that used stem cells.  36% of these trials were in the United States, 17% of them were in China, 8% in Japan, 5% in Spain, just under 5% were in India, 3.5 % were in South Korea and Iran, and 2% were in the UK.  To further break down these numbers according to geographical region, 36% were in the North America, 35% were in Asia, 19% were in Europe, 5% were in the Middle East, 3% were in Central and South America, and 2% were in Australia.

Of these clinical trials, 116 used mesenchymal stem cells, 81 used T-Cells, 31 used dendritic cells, 26 used mononuclear cells from bone marrow, 10 used Natural Killer cells, 22 used stromal vascular fraction (SVF) cells from fat, 16 used HSPCs (hematopoietic and progenitor cells) from bone marrow, and three were embryonic stem cell trials.

What were these trials trying to treat?  123 were for cancers of some sort, there were 51 trials examining neurological diseases and also 51 trials examining musculoskeletal disorders, 26 trials trying to help people with cardiovascular diseases, 17 attempting to treat skin diseases, 15 treating eye diseases, 8 that treated liver diseases, and 5 diabetes trials.

These are the rough trends.  As you can see, clinical trials that utilize adult and umbilical stem cell stem cells VASTLY outnumber those that use embryonic stem cells.

Bersenev Alexey. Trends in cell therapy clinical trials 2011 – 2014. CellTrials blog. February 14, 2015. Available: http://celltrials.info/2015/02/14/trends-2014/

Stem Cell Treatments for Diabetic Retinopathy


Research by a team at the University of Virginia School of Medicine provided a crucial piece to treating patients who suffer vision loss because of diabetic retinopathy, a condition that affects millions of people with diabetes. The UVA team showed that the best for adult stem cells to treat this condition are cells taken from donors who do not suffer diabetes rather than cells taken from patients’ own bodies. This work could provide a critical step toward injecting stem cells into patients’ eyes to stop or even reverse vision loss. These findings could also establish a crucial framework for evaluating stem cells to be used in potential future treatments for diabetic retinopathy.

Diabetic Retinopathy

“It answers a vital question: If we’re going to carry this therapy forward into clinical trials, where are we going to get the best bang for the buck?” said UVA researcher and ophthalmologist Paul Yates, M.D., Ph.D. “The answer seems to be, probably, taking cells from patients who aren’t diabetic. Because the diabetic stem cells don’t seem to work quite as well. And that’s not terribly surprising, because we already know that this cell type is damaged by diabetes.”

The researchers hope to use stem cells derived from fat, since they are harvested during liposuction procedures. These fat-based stem cells might be able to stop or greatly delay the vascular degeneration that eventually leads to blindness in patients with diabetic retinopathy. What are the best cells for the job in this case? UVA’s new research provides those important answers. “We now know what to look for when we harvest a patient’s cells, because we know what distinguishes good quality cells from poor quality,” said researcher Shayn M. Peirce, Ph.D., of the UVA Department of Biomedical Engineering. “We almost have a screen to determine quality control. We’re essentially establishing quality-control criteria by understanding what works and why.”

Diabetic retinopathy patients desperately need new and more effective treatments. First of all, there are a growing number of people with this condition and secondly the present treatments only show limited effectiveness. More than 100 million people are estimated to suffer from diabetic retinopathy and related conditions; current treatments use lasers to fight back invading blood vessels, but these treatments often destroy much of the retina. Alternatively, patients are required to receive injections of anti-blood vessel forming drugs such as Lucentis (ranibizumab) or Eylea (aflibercept) directly into their eyeball, sometimes every month, for the rest of their lives.

“There’s huge room for improvement on the standard of care, and the number of patients in this demographic is increasing by the day, dramatically, so the need is only going up,” Peirce said. “So I think there are three pieces working together — UVA’s strengths in this area, the FDA’s encouragement [of stem cell research in the eye] and the clinical realities — to drive this cell-based therapy toward the clinic.”

While much more work needs to be done, if all goes well, the UVA team hopes to begin clinical trials in humans within the next few years. “This is not science fiction at all,” Yates said. “The idea that you can take cells from somewhere else and inject them into the eye to treat disease is here today.”

University of Pittsburgh Team Uses Patient’s Own Stem Cells to Clear Cloudy Corneas


The transparent portion of the center of our eyes is called the cornea. Scars on the cornea can cause an infuriating haziness across the eye. However, healing these cloudy corneas might be as simple as growing stem cells from a tiny biopsy of the patient’s undamaged eye and placing them on the injury site. This hope comes from experiments in a mouse model system conducted by researchers at the University of Pittsburgh School of Medicine. These findings were published in Science Translational Medicine and could one day rescue vision for millions of people worldwide and decrease the need for corneal transplants.

According to statistics compiled by the National Eye Institute, which is a branch of the National Institutes of Health, globally, corneal infectious diseases have compromised the vision of more than 250 million people and have blinded over 6 million of them. Additionally, trauma from burns is also a leading cause of corneal scarring.

James L. Funderburgh, Ph.D., professor of ophthalmology at Pitt and associate director of the Louis J. Fox Center for Vision Restoration of UPMC and the University of Pittsburgh, a joint program of UPMC Eye Center and the McGowan Institute for Regenerative Medicine, said, “The cornea is a living window to the world, and damage to it leads to cloudiness or haziness that makes it hard or impossible to see. The body usually responds to corneal injuries by making scar tissue. We found that delivery of stem cells initiates regeneration of healthy corneal tissue rather than scar leaving a clear, smooth surface.”

The lead author of this study, Sayan Basu, is a corneal surgeon who works at the L.V. Prasad Eye Institute in Hyderabad, India. Dr. Basu who joined with Dr. Funderburgh’s lab, has developed a technique to isolate ocular stem cells from tiny biopsies from the surface of the eye and a region between the cornea and sclera known as the limbus. Such a small biopsy heals rapidly with little discomfort and no disruption of vision. Such biopsies are banked in tissue banks and then expanded in culture, and several tests shows that even after isolation and expansion, these cells are still corneal stem cells.

limbal-stem-cells

“Using the patient’s own cells from the uninjured eye for this process could let us bypass rejection concerns,” Dr. Basu noted. “That could be very helpful, particularly in places that don’t have corneal tissue banks for transplant.”

Basu in collaboration with Funderburgh’s team tested these human limbal stem cells in a mouse model of corneal injury. This team used goo made of fibrin to glue the cells to the injury site. Fibrin is the protein found in blood clots, but it is also commonly used as a surgical adhesive. Application of these limbal stem cells not only induced healing of the mouse corneas, their eyes became clear again within four weeks of treatment. On the other hand, the eyes of mice that were not treated with limbal stem cells remained cloudy.

Fibrin

In fact, the healing was so good that Funderburgh said: “Even at the microscopic level, we couldn’t tell the difference between the tissues that were treated with stem cells and undamaged cornea. We were also excited to see that the stem cells appeared to induce healing beyond the immediate vicinity of where they were placed. That suggests the cells are producing factors that promote regeneration, not just replacing lost tissue.”

This work is the impetus behind a small pilot study presently underway in Hyderabad which will treat a handful of patients with their own corneal stem cells.

The First Patient Treated with iPSC-Derived Cells


Nature News has reported that a Japanese patient was received the first treatment derived from induced pluripotent stem cells.

Ophthalmologist Masayo Takahashi from the Riken Center for Developmental Biology and her team used genetic engineering techniques to reprogram skin fibroblasts from this patient into induced pluripotent stem cells. These cultured iPSCs were then differentiated into retinal pigment epithelium cells. Takahashi’s colleagues, led by Yasuo Kurimoto at Kobe City Medical Center General Hospital, then implanted those retinal pigment epithelium cells into the retina of this female patient, who suffers from age-related macular degeneration.

It is unlikely that this procedure will restore the woman’s vision. However, because age-related macular degeneration is a progressive process, Takahashi and her research team will be examining if this procedure prevents further deterioration of her sight. Takahashi’s Riken team has extensively tested this procedure in laboratory animals and recently received human trial clearance. Takahashi’s team will also be looking particularly hard at the side effects of this procedure; such as immune reaction or cancerous growth.

“We’ve taken a momentous first step toward regenerative medicine using iPS cells,” Takahashi says in a statement, according to Nature News. “With this as a starting point, I definitely want to bring [iPS cell-based regenerative medicine] to as many people as possible.”

Induced Pluripotent Stem Cells Form Limbal-Like Stem Cells


Limbal epithelial stem cells or LESCs are found at the periphery of the cornea and they continuously renew the corneal epithelium. Loss of this stem cell population can cause loss of corneal transparency and eventual loss of vision.

Genetic conditions can cause LESC deficiency, such as congenital aniridia, Stevens-Johnson syndrome or Ocular cicatricial pemphigoid. Other causes of LESC deficiency include chemical or thermal burns to the eye, microbial infections, extended contact lens wear, sulfur mustard gas poisoning, or chronic inflammation of the eye,

Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman's layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.
Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman’s layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.

Treatments of LESC deficiency include limbal stem cell grafts from one eye to another, but these grafts have a 3-5-year graft survival of only 30%-45%. If LESCs are expanded in culture on human amniotic membrane, then 76% of the grafts will successfully take 1-3 years after grafting. This procedure is not standardized. If LESCs are grafted from a cadaver, their survival is low.

Given these less than optimal treatments for LESC deficiencies, Alexander Ljubimov and his team from UCLA have used induced pluripotent stem cells (iPSCs) to make cultured LESCs. Ljubimov and his coworkers derived iPSCs from the skin cells of volunteers with non-integrating plasmids. Then they grew these cells on corneas that have been stripped of their cells and human amniotic membranes and these cells differentiated into LESC-like cells.

Ljubimov and others also made iPSCs from human LESCs, and when they cultured these iPSCs derived from LESCs on human amniotic membranes for two weeks, the cells differentiated into LESCs that made LESC-specific genes, and had the epigenetic characteristics of LESCs.

These experiments show that the cell source for iPSC derivation can greatly influence the epigenetic characteristics of the iPSC line. Also these experiments show that iPSCs can be used to make LESCs that can potentially be used for therapeutic purposes.

Patient-Specific Stem Cells Plus Personalized Gene Therapy for Blindness


Researchers from Columbia University Medical Center (CUMC) have devised protocols to develop personalized gene therapies for patients with an eye known as retinitis pigmentosa (RP), which is a leading cause of vision loss. While RP can begin during infancy, the first symptoms typically emerge during early adulthood. Typically the disease begins with night blindness, and RP eventually progresses to rob the patients of their peripheral vision. In its later stages, RP destroys photoreceptors in the macula, that region of the retina that provides the best vision under lighted conditions. RP is estimated to affect at least 75,000 people in the United States and 1.5 million worldwide.

The approach utilized by this Columbia team utilizes induced pluripotent stem (iPS) cell technology to transform patient’s skin cells into retinal cells, which are then used as a patient-specific model for disease study and preclinical testing.

The leader of this research group, Stephen H. Tsang, MD, PhD, showed that a form of RP caused by mutations to the MFRP gene compromised the structural integrity of the retinal cells. The MFRP gene encodes a protein called the Membrane Frizzled-Related Protein, which plays an important role in eye development. Mutations in the MFRP gene are associated with small eye conditions such as nanophthalmos, posterior microphthalmia, or retinal issues such as retinitis pigmentosa, foveoschisis, or even optic disc drusen. Tsang and his group, however, showed that the effects of these MFRP mutations could be reversed with gene therapy. Thus this new approach could potentially be used to create personalized therapies for other forms of RP, or even other genetic diseases.

“The use of patient-specific cell lines for testing the efficacy of gene therapy to precisely correct a patient’s genetic deficiency provides yet another tool for advancing the field of personalized medicine,” said Dr. Tsang, the Laszlo Z. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology. This work was recently published in the online edition of Molecular Therapy, the official journal of the American Society for Gene & Cell Therapy.

Mutations in more than 60 different genes have been linked to RP. Such a genetic disease is known as a heterogeneous trait and genetic diseases like RP or deafness or other such conditions are very difficult to develop models to study. Animal models, though useful, have significant limitations because of interspecies differences. Eye researchers have also used human retinal cells from eye banks to study RP. This eye tissue comes from the eyes of patients who suffered from the disease and donated their eye tissue to research after death. Unfortunately, despite their usefulness, donated eye tissues typically illustrate the end stage of the disease process. Despite their usefulness, they reveal little about how RP develops. Also, there are no human tissue culture models of RP, since it is dangerous to harvest retinal cells from patients. Finally, human embryonic stem cells could be useful in RP research, but they are fraught with ethical, legal, and technical issues.

However, the Tsang group used iPS technology to transform skin cells from RP patients, each of whom harbored a different MFRP mutation, into retinal cells. Thus they created patient-specific models for studying the disease and testing potential therapies. Because they used iPS technology, Tsang found a way around the limitations and concerns and dog embryonic stem cells. Thus researchers can induce the patient’s own skin cells and de-differentiated them to a more basic, embryonic stem cell–like state. Such cells are “pluripotent,” which means that they can be transformed into specialized cells of various types.

When Tsang and others analyzed these patient-specific cells, they discovered that the primary effect of MFRP mutations is to disrupt the regulation of a cytoskeletal protein called actin, the scaffolding that gives the cell its structural integrity. “Normally, the cytoskeleton looks like a series of connected hexagons,” said Dr. Tsang. “If a cell loses this structure, it loses its ability to function.” They also found that MFRP works in tandem with another gene, CTRP5, and that a balance between the two genes is required for normal actin regulation.

In the next phase of the study, the CUMC team used adeno-associated viruses (AAVs) to introduce normal copies of MFRP into the iPS-derived retinal cells. This successfully restored the cells’ function. Tsang and others used gene therapy to “rescue” mice with RP due to MFRP mutations. According to Dr. Tsang, the mice showed long-term improvement in visual function and restoration of photoreceptor numbers.

“This study provides both in vitro and in vivo evidence that vision loss caused by MFRP mutations could potentially be treated through AAV gene therapy,” said coauthor Dieter Egli, PhD, an assistant professor of developmental cell biology (in pediatrics) at CUMC, who is also affiliated with the New York Stem Cell Foundation.

Dr. Tsang thinks this approach could potentially be used to study other forms of RP. “Through genome-sequencing studies, hundreds of novel genetic spelling mistakes have been associated with RP,” he said. “But until now, we’ve had very few ways to find out whether these actually cause the disease. In principle, iPS cells can help us determine whether these genes do, in fact, cause RP, understand their function, and, ultimately, develop personalized treatments.”

A New Way to Regrow Human Corneas


My apologies to my readers, but I was at the Free Methodist Bible Quizzing Finals at Greenville College in Illinois for the last week. I am recovering and have only the energy to write a short post for today.

The cornea is the transparent covering of the eye that transmits light from the environment to the inside of the eye, to the photoreceptor-rich retina that interprets the light information and translates them into neural signals that are sent to the visual centers of the brain.

The cornea is subject to constant wear and tear, but fortunately, a stem cell population called limbal stem cells. These stem cells constantly regenerate the cornea, and the conjunctiva, which is otherwise known as the “whites of the eye.”

Limbal stem cells

 

Unfortunately, the limbal stem cells can be damaged by chemicals, sparks from a welding, genetically inherited conditions, or physical trauma. Such conditions can prevent proper replacement of constantly sloughed cornea and conjunctival cells. This can seriously compromise the structural integrity and function la of the eye.

To treat patients with corneal limbal stem defects, eye surgeons have transplanted limbal cells from cadavers or used small excisions of limbal cell populations from the unaffected eye and transplanted them into the affected eye. These so-called “autologous limbal cell transplants” tend to work quite well, but there are two cuts that need to be made. Is there are way to expand limbal stem cells for clinical use? Now it appears that there is.

Scientists from the Massachusetts Eye and Ear Infirmary have used sophisticated key tracer molecules to pin down the precise cells in the eye that are capable of regeneration and repair. They then transplanted these regenerative stem cells into mice to create fully functioning corneas.

This work was published in the international journal Nature, and they predict that this method may one day help restore the sight of victims of burns and chemical injuries.

Limbal stem cells (LSC) completely renew our corneas every few weeks and repair the cornea and conjunctiva whenever they are injured. Without LSCs the cornea becomes cloudy, which severely disrupts vision. In fact, LSC deficiencies are among the commonest reasons behind blindness worldwide.

Unfortunately, the LSC population is embedded in a part of the eye where they share space with a matrix of other structures in the limbal part of the eye (FYI – the limbus is the junction between the cornea and the white of the eye).

Enter the work from the Massachusetts Eye and Ear Infirmary in Boston at the Boston Children’s Hospital, Brigham and Women’s Hospital and in collaboration with the VA Boston Healthcare System have identified a key molecule known as ABCB5, which is naturally present on the surface of LSCs.

Although ABCB5 has been known about for some time in other parts of the body, this is the first time ABCB5 has been identified on the surfaces of LSCs. Also, it is clear that ABCB5 can effectively mark LSCs.

By using molecules linked to fluorescent molecules, these scientists were able to instantly identify a pool of LSCs on donated human corneas. After transplanting these cells into the eyes of mice, they discovered that the transplanted cells were able to generate fully functioning human corneas.

Prof Markus Frank, of Boston Children’s Hospital, a lead author in the research, told the BBC: ” The main significance for human disease is we have established a molecularly defined population of cells that we can extract from donor tissue.

“And these cells have the remarkable ability to self-regenerate. We hope to drive this research forward so this can be used as a therapy.”

Harminder Dua, professor of ophthalmology at the University of Nottingham, who was not involved in this study, said: “This paper represents a very comprehensive and well conducted piece of work that takes use closer to the precise identification of stem cells.

“Applying this knowledge to a clinical setting could help improve the outcomes for patients who need corneal reconstruction.”

Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection


Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.

Treating Age-Related Blindness with a Stem Cell Replacement Method


A collaboration between German and American scientists in New York City has resulted in the invention of a new method for transplanting stem cells into the eyes of patients who suffer from age-related macular degeneration, which is the most frequent cause of blindness. In an animal test, the implanted stem cells survived in the eyes of rabbits for several weeks.

Approximately 4.5 millino people in Germany suffer from age-related macular degeneration (AMD), which causes gradual loss of visual acuity and affects the ability to read, drive a car or do fine work. The center of the vision field becomes blurry as though covered by a veil. This vision loss is a consequence of the death of cells in the retinal pigment epithelium or RPE, which lies are the back of the eye, underneath the neural retina.

Inflammation within the RPE causes AMD. Increased inflammation prevents efficient recycling of metabolic waste products, and the build-up of toxic wastes causes RPE die off. Without the RPE, the photoreceptors in front of the RPE cells that also depend on the RPE to repair the damage suffered from continuous light exposure, begin to die off too.

RPE

Retinal Pigmented Epithelium

Presently no cure exists for AMD, but scientists at Bonn University, in the Department of Ophthalmology and New York City have tested a new procedure that replaces damaged RPE cells.

In the present experiment, RPE cells made from human stem cells were successfully implanted into the retinas of rabbits.

Boris V. Stanzel, the lead author of this work, said, “These cells have now been used for the first time in research for transplantation purposes.”

The adult RPE stem cells were characterized by Timothy Blenkinsop and his colleagues at the Neural Stem Cell Institute in New York City. Blenkinsop designed methods to isolate and grow these cells. He also flew to Germany to assist Dr. Stanzel with the transplantation experiments.  Blenkinsop obtained his RPE cells from human cadavers, and he grew them on polyester matrices.

These experiments demonstrate that RPE cells obtained from adult stem cells can replace cells destroyed by AMD. This newly developed transplantation method makes it possible to test which stem cells lines are most suitable for transplantation into the eye.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

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.

Treating Diabetic Retinopathy with Stem Cells


Scientists at Queen’s University Belfast hope to design a new approach for treating the eyesight of diabetic patients by using adult stem cells.

Millions of diabetics every year are at risk for losing their eyesight due to diabetic retinopathy. When high blood sugar causes blood vessels in the eye to leak or become blocked, failed blood flow damages the retina and lead to vision impairment. If left untreated, diabetic retinopathy can lead to blindness.

The Queen’s University Belfast group have initiated the REDDSTAR study, which stands for Repair of Diabetic Damage by Stromal Cell Administration, and this study involves researchers from the Queen’s Center for Vision and Vascular Science in the School of Medicine, Dentistry and Biomedical Sciences. REDDSTAR begins with the isolation of stem cells from patients and expanding them in the laboratory. Then these patient-specific cells are delivered to the patient from whom they were originally drawn in order to repair the blood vessels in the eye. This blood vessel repair is especially useful in patients with diabetic retinopathy.

Presently, diabetic retinopathy is treated with laser ablation of new blood vessels that grow in response to damage. These new blood vessels become so dense that they obscure vision. However, presently, there are no treatments to control the progression of diabetic complications.

Alan Stitt, the director of the Centre for Vision and Vascular Science at Queen’s and lead scientist for the REDDSTAR study, said, “The Queen’s component of the REDDSTAR study involves investigating the potential of a unique stem cell population to promote repair of damaged blood vessels in the retina during diabetes.” Professor Stitt continued: “The impact could be profound for patients, because regeneration of damaged retina could prevent progression of diabetic retinopathy and reduce the risk of vision loss.”

“Treatments for diabetic retinopathy are not always satisfactory. They focus on end-stages and fail to address the root causes of the condition. A novel, alternative therapeutic approach is to harness adult stem cells to promote regeneration of the damaged retinal blood vessels and thereby prevent and/or reverse retinopathy.”

Stitt said the new research project is one of several regenerative medicine approaches ongoing at his research center. Their approach is to isolate a rather well-defined population of stem cells and then deliver those stem cells to sites in the body that have been ravaged by diabetes. In particular patients, these strategies have produced remarkable benefits from stem cell-mediated repair of their blood vessels. Treatments such as this one are simply the first step in the quest to develop exciting, effective and new therapies in an area of medicine where such therapies are desperately needed.

In the REDDSTAR study, stem cells from bone marrow are used and these stem cells are provided by Orbsen Therapuetics, which is a spin-off from the Science Foundation Ireland-funded Regenerative Medicine Institute (REMEDI) at NUI Galway.

This project will design protocols for growing these bone marrow-derived stem cells and they will be tested in several preclinical models of diabetes and diabetic complications at research centers in Belfast, Galway, Munich, Berlin, and Porto before human clinical trails take place in Denmark.

Queen’s Centre for Vision and Vascular Science is a key focus of the University’s ambitious 140-million pound “together we can go Beyond” fundraising campaign. This campaign is due to expand the Vision Science program further when the University’s new 32-million pound Wellcome-Wolfson Centre for Experimental Medicine opens in 2015. Along with vision, two new programs in Diabetes and Genomics will also be established in the new Center. These Center should stimulate further investment and global collaborations between biotech and health companies in Ireland.

Stem Cell Treatments for Retinitis Pigmentosa Inch Toward Clinical Trials


Retinitis Pigmentosa or RP is the most common form of inherited blindness. There are many different genes involved in the onset of RP. Molecular defects in more than 40 different genes can cause “isolated RP” and defects in more than 50 different genes can cause “syndromic RP.” Not only are there a host of different genes involved in RP, two patients with exactly the same molecular lesion can have a type of RP that differs substantially in its presentation.

The retina at the back of the eye is composed of two thick layers known as the inner neural retina and the outer pigmented retina. The neural retina consists of an outer layer of photoreceptors that are connected to an inner layer of bipolar cells. The bipolar cells connect with ganglion cells that have axons that extend to the optic nerve. The photoreceptor cells have their tips embedded in the pigmented retina, and the pigmented retina maintain and nourish the photoreceptors.

Pigmented Retina

If the pigmented retina does not function properly, then the effects are most profoundly displayed in the photoreceptors. Photoreceptors respond to light and the constant exposure to light causes the photoreceptors to take a beating. The byproducts of all that light-induced damage accumulates at the tips of the photoreceptors cells, and these rubbish-filled tips are taken a gulped down by the cells of the pigmented retina. The pigmented retina cells degrade the damaged byproducts and recycle the precursor molecules. Without properly functioning pigmented retina cells, the photoceptors cells accumulate toxic light damage and then eventually die. Photoreceptor cell death is the end product of RP, and it results in blindness.

There is no cure for RP, and the treatments available are very hit-and-miss. For this reason, cell therapies have been examined in a variety of animal models of RP, which, in many cases, closely mimic the human disease to some degree.

Two different experimental treatments, one with induced pluripotent stem cells (iPSCs) and another with gene therapy have produced long-term improvement in visual function in mice with RP. These studies have been conducted at the Columbia University Medical Center (CUMC).

Stephen Tsang, associate professor of pathology, cell biology and ophthalmology who led both studies commented: “While these therapies still need to be refined, the results are highly encouraging. We’ve never seen this type of improvement in retinal function in mouse models of RP. We hope we may finally have something to offer patients with this form of vision loss.”

In one study, CUMC researchers tested the long-term safety and efficacy of iPSC grafts into the pigmented retina to restore visual function in a mouse model of RP. The mice were injected with undifferentiated iPSCs when they were five years old, and the cells differentiated into retinal pigmented epithelial (RPE) cells and integrated into the retinas. None of the mice that received these transplantations developed tumors over their lifetimes.

To test the effects of the implanted cells on the vision of the mice, Tsang’s group used electrophysiological measurements of the retina. In RP mice, as they become blind, the electrophysiology of the retina becomes rather abnormal, but in these mice implanted with the iPSCs, the electrophysiology of their retinas were not only normal, but stayed normal for a long period of time.

According to Tsang: “This is the first evidence of lifelong neuronal recovery in an animal model using stem cell transplants, with vision improvement persisting throughout the lifespan.”

In 2011, the FDA approved clinical trials of embryonic stem cell (ESC) transplants for the treatment of macular degeneration, but this treatment requires the application of drugs that suppress the immune system. Such drugs have rather nasty side effects.

“Our study focused on patient-specific iPS cells, which offer a compelling alternative,” Tsang said. “The iPS cells can provide a potentially unlimited supply of cells for functional rescue and optimization. Also, since they would come from a patient’s own body, immunosuppression would not be necessary to prevent rejection after transplantation.”

Theoretically, iPSC transplants, could also be used to treat age-related macular degeneration, which is the leading cause of vision loss in older adults.

In a second approach to treating RP, CUMC scientists tested a gene therapy protocol in RP mice. A specific type of RP that results from mutations in a PDE6alpha gene was used as a model system for gene therapy protocol. This particular type of RP is rather common in humans. The CUMC scientists injected a virus into one of the eyes of afflicted mice. This virus was engineered to express the PDE6alpha gene when it entered cells. Because this virus is the AAV or adenovirus-associated virus, it only spreads in the presence of adenovirus. Without a helper adenovirus in the retina, the engineered virus particles will infect the cells they initially contact, but they will not produce a productive infection. However, ferry the genes inside them to the cell they initially infect. This the engineered AAV particles are excellent vehicles for getting genes inside cells without causing an infection.

Examination of the mice six months later, the photoreceptors in the AAV-treated eyes were healthy and these eyes were able to see, but the uninjected eyes were unable to see and their photoreceptors were mostly dead.

Again Tsang commented: “These results provide support that RP due to PDE6alpha deficiency in humans is also likely to be treatable by gene therapy.”

CUMC and its teaching-hospital affiliate, New York-Presbyterian Hospital are part of an international consortium that was recently formed to bring this PDE6A gene therapy to patients. Pending FDA approval, clinical trials could begin within a year.

See  Li, Y., Tsai, Y.T., Hsu, C.W., Erol, D., Yang, J., Wu, W.H., Davis, R.J., Egli, D., and Tsang, S.H. Long-term safety and efficacy of human induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa. Mol Med. 2012 Aug 9. doi: 10.2119/molmed. 2012.00242. [Epub ahead of print] (2012).

Wert KJ, Davis RJ, Sancho-Pelluz J, Nishina PM, Tsang S.H. Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa. Hum. Mol. Genet. (2012) doi: 10.1093/hmg/dds46

A New Technique to Fix Damaged Eyes With Stem Cells


Engineers at the University of Sheffield have invented a new delivery technique for delivering stem cells to eyes. They have high hopes that this technique will help repair the eyes of those patients who have suffered damage to their eyes.

The front of the eye is bordered by the transparent cornea, which transmits light to the lens. The cornea is exposed to the outside world and if there is an accident that affects the eye, the cornea is usually the part that takes a beating. The cornea undergoes constant turnover as dead cells are constantly sloughed from the cornea during blinking. At the junction between the cornea and the sclera is an area called the limbus. Located at the limbus is a population of limbal epithelial stem cells or LESCs. LESCs have many features commonly observed in other stem cells, such as small size, high nuclear to cytoplasmic ratio, and they lack expression of molecules commonly found in mature corneal cells, such as cytokeratins 3 and 12.

Human Limbus

LESCs are slow-growing, but in the event of injury they can become highly proliferative (See Lavker R.M, Sun T.T. Epithelial stem cells: the eye provides a vision. Eye. 2003;17:937–942. DOI: 10.1038/sj.eye.6700575).

LESC deficiency can result from chemical or thermal burns to the eye or as a result of certain inherited diseases. Partial or full LESC deficiency causes abnormal corneal wound healing and surface integrity. Also LESC deficiency causes the conjunctiva to grow over the cornea, and this is disastrous for the eye because the cornea is devoid of blood vessels, which is the reason why it is transparent. However the conjunctiva (the white of the eye) is filled with blood vessels and is not transparent. Thus chronic inflammation, recurrent erosion, ulceration and stromal scarring can occur and cause painful vision loss

Long term restoration of visual function requires renewal of the corneal epithelium, and this requires the placement of a new stem cell population by means of a limbus graft. From where do you get a new limbus for transplantation? Autografts use limbal cells from the good eye, but this runs the risk of scarring the cornea of the other eye.procedure is the use limbal cells from cadavers (limbal allografts). Also, making sure that the graft adheres to the requires the use of sutures, but these sutures can cause substantial amounts of irritation. Therefore, the Sheffield research group designed a new technique.

With this new technique, a disk made of biodegradable material is loaded with limbal stem cells and then placed over the eye. This disc has an outer ring pockmarked with small niches for stem cells can hide. The material in the center of the disc is thinner than that on the edges, and therefore, the center of the disc biodegrades faster. This releases the stem cells in center of the disc into the cornea where they can grow and help repair it.

Because these small niches in the disc resemble the stem cells niches found in the limbus, these discs do an excellent job of nurturing the limbal stem cells and distributing them to the cornea. Limbal grafts are either done with amniotic membrane as a carrier, but this procedure leads to increased inflammation in the eye and there is a chance that the grafts will not integrate into the limbus. The biodegradable disc groups the limbal stem cells into clusters that are more likely to ingrate into the limbus.

According to Professor Sheila MacNeil, “Laboratory tests have shown that the membranes will support cell growth, so the next stage is to trial this in patients in India, working with our colleagues in the LV Prasad Eye Institute in Hyderabad. One advantage of our design is that we have made the disc from materials already in use as biodegradable sutures in the eye so we know they won’t cause a problem in the body. This means that, subject to the necessary safety studies and approval from Indian Regulatory Authorities, we should be able to move to early stage clinical trials fairly quickly.”

In the developing world, corneal blindness is rather common in some professions and treating it is a rather pressing problem. High instances of chemical burns to the eye or accidental damage to the eye are common, but complex treatment strategies such as amniotic membrane grafts are not available to the general public.

This technique also possibilities in more developed countries, since current techniques use donor tissue to deliver the cultured cells, and this requires a tissue bank to which some people do not have access. Also, the use of the cell-impregnated disk will reduce the risk of disease transmission with grafts.

StemCells Inc. Announces the Commencement of Their Macular Degeneration Clinical Trial


Age-related macular degeneration or AMD is a disease of the retina (at the back of the eye) characterized by a loss of photoreceptors (rods and cones) from the central part of the retina (macula), where vision in the clearest. A degenerative retinal disease, AMD typically strikes adults in their 50s or early 60s, and insidiously progresses usually painlessly until it gradually destroys central vision. There are approximately 1.75 million Americans age 40 years and older with some form of AMD, and the disease continues to be the number one cause of irreversible vision loss among senior citizens in the United States with more than seven million at risk of developing AMD.

There are no cures for AMD, but laser treatments are available for some types of AMD. Laser photocoagulation can disperse fluid that has built up under the retina. Such AMD is called “wet” macular degeneration and only works in the treatment of 15/100 cases of AMD. Other treatments include injections of either Avastin, Macugen or Eylea into the eye to prevent the spread of blood vessels that crowd out photoreceptors.  Photodynamic therapy uses a drug called Visudyne that is injected into the arm and them activated by a laser one in the eye where it destroys meandering blood vessels that leak or proliferate across the retina.  Patients with “dry” macular degeneration, however, find themselves out of luck.

Into the breach comes a clinical study by StemCells Inc. to use their proprietary neural stem cell line to treat dry macular degeneration. This Phase I/II clinical trial has already enrolled and transplanted its first patient this week and more subjects will undoubtedly be enrolled later. This trial is designed to evaluate the safety and preliminary efficacy of StemCells Inc’s proprietary HuCNS-SC neural stem cell line as a treatment for dry AMD. The first patient in this clinical trial received their transplant at the Retina Foundation of the Southwest in Dallas, Texas, which is one of the leading independent vision research centers in the United States. Globally, AMD afflicts approximately 30 million people worldwide and is the leading cause of vision loss and blindness in people over 55 years of age.

In February 2012, StemCells Inc Company published preclinical data that clearly showed that HuCNS-SC cells protect host photoreceptors and preserve vision in the rats that are engineered to experience retinal degeneration. This rat strain (Royal College of Surgeons or RCS rats) are a very well-established animal model for retinal disease and has been used extensively to evaluate potential cell therapies. In these pre-clinical studies, the number of cone photoreceptors, which are responsible for central vision, did not decrease due to cell death, but instead remained constant over an extended period. These same rats that had HuCNS-SC cells transplanted into their retinas showed steady maintenance of their visual acuity and light sensitivity. In humans, degeneration of the cone photoreceptors accounts for the unique pattern of vision loss in dry AMD. These data were published in an international peer-reviewed journal known as the European Journal of Neuroscience.

“This trial signifies an exciting extension of our on-going clinical research with neural stem cells from disorders of the brain and spinal cord to now include the eye,” said Stephen Huhn, MD, FACS, FAAP, Vice President and Head of the CNS Program at StemCells, Inc. “Studies in the relevant animal model demonstrate that the Company’s neural stem cells preserve vision in animals that would otherwise go blind and support the therapeutic potential of the cells to halt retinal degeneration. Unlike others in the field, we are looking to intervene early in the course of the disease with the goal of preserving visual function before it is lost.”

David G. Birch, Ph.D., Chief Scientific and Executive Officer of the RFSW and Director of the Rose-Silverthorne Retinal Degenerations Laboratory and principal investigator of the study, added, “We are excited to be working with Stem Cells [Inc.} on this ground breaking clinical trial. There currently are no effective treatments for dry AMD, which is the most common form of the disease, and there is a clear need to explore novel therapeutic approaches.”

Using Cultured Limbal Cells from Cadavers to Heal Blindness


Stem cell-based therapies have been available for the eye for several years. In particular, diseases of the outermost layer of the eye, the cornea, can be treated with “limbal cell” transplantations.

The human eye is more or less spherical, but is rather asymmetrical. Our eyes are also one inch in diameter. The eye consists of a front and rear compartment. The front compartment consists of the iris, which is pigmented, the cornea, which is transparent, the pupil, which is the black, round opening in the iris that lets light in, the sclera or white part of the eye, and the conjunctiva, which is an invisible, clear layer of tissue that covers the front of the eye, which the exception of the cornea. Just behind the iris and the pupil lies the lens, which focuses the light on the back of the eye. Most of the eye is filled with a clear gel called vitreous.

The rear compartment is filled with vitreous humor, which is a liquid that is also rich in a slippery, acidic carbohydrate called hyaluronic acid and several types of proteins. The back of the eye is covered with special light-sensing cells that are collectively called the retina. The retina converts the energy from the visible spectra of light into electrical impulses, and behind the eye, the optic nerve carries these impulses to the brain. The macula is a small, sensitive area within the retina that gives central vision. It is located in the center of the retina and contains the fovea, a small depression or pit at the center of the macula that gives the clearest vision.

If we now focus on the cornea, we will see that there is a ring of tissue that connects the cornea and the sclera known as the limbus. The limbus possesses a stem cell population that replenishes the cornea and also serves as a barrier for the cells on the conjunctiva. If this stem cell population is damaged or depleted, then conjunctival cells invade the cornea, and disrupt its unique structure. The cornea is transparent and allows light to pass through it unperturbed. The conjunctiva, however, is translucent, and filled with blood vessels. If the cornea becomes invaded with conjunctiva cells, it will cloud over and vision will be obscured.

To fix this problem, scientists and eye surgeons have experimented with limbal stem cell transplants. The most successful forms of transplantation use limbal cells from one eye that are transplanted into the other eye. This procedure, however, has a few drawbacks. The removal of limbal cells from one eye can compromise the integrity of the donor eye. Secondly, the patient is now left with two eyes that are healing rom surgery.

A second procedure uses limbal cells from cadavers. This procedure provides a better solution, but the availability of the tissue is a problem. To solve the problem of insufficiency of tissue, several labs have tried to culture the limbal cells and grow them to larger quantities.

A paper in the British Journal of Ophthalmology by Basu and others from the Prasad Eye Institute in Hyderbad, India has examined many patients who received limbal cell transplantations from cadavers. They followed 28 eyes from 21 patients who suffered from limbal stem cell deficiency (LSCD). While this disease is relatively rare, it prevents patients from receiving limbal cell grafts from their own eyes, since both eyes are deficient for limbal stem cells. These patients were treated between 2001 and 2010, and all limbal cells were cultured in the laboratory first and then transplanted 10-14 days after their removal from the cadaver.

Each patient was followed up after surgery for about 5 years, and 71.4% of all patients showed eyes that were stable and clear. The eye sight in the treated eye improved to 20/60 or better in 19 of the 28 treated eyes. There were no serious ocular complications in any patients.

This paper shows that transplantation of cultured limbal cells from cadavers successfully restores the surface of the eye and improves vision in patients with blindness as a result of LSCD. This same technique can also be applied to patients with other types of eye surface disorders. Limbal stem cell transplantations seem to keep improving and they will hopefully become rather routine.

Human-Eye Precursors are Grown from Embryonic Stem Cells


Yoshiki Sasai of the RIKEN Center for Developmental Biology (CBD) in Kobe, Japan has managed to grow eye precursors in the laboratory from embryonic stem cells.  Such an achievement provides a remarkable opportunity to investigate early eye development and the pathology of eye abnormalities.

Eye development is a complex process, since mammalian eyes develop as an extension of the central nervous system.  The development of the central nervous system begins at about 18 days after fertilization with the formation of a thickened layer of cells on the surface of the embryo called the neural plate.  The neural plate is induced by a cluster of cells that clumps together to form a hollow tube called the “notochordal plate.”  The neural plate rolls into a tube called the neural tube and this neural tube is the beginnings of the central nervous system.  The front of the neural tube will inflate to form the brain and the portion of the tube behind the brain forms the spinal cord.  The neural tube forms as a result of high points that form in the neural plate called the neural folds.  These neural folds fuse to form a tube that is below the outermost layer of the embryo (ectoderm).

About 22 days after fertilization, inflations on either side of the developing brain extend from the brain, and these are the beginnings of the eye.  These “optic vesicles” as they are called continue to grow until their connection to the brain becomes narrower and narrower.  The narrow connections between the optic vesicles and the brain are called the optic stalks and they will become the rudiments of the optic nerves.

The optic vesicles make contact with the surface of the embryo and this does two things.  The vesicle collapses into a two-layered structure called the optic cup and the embryonic ectoderm pinches in and forms a vesicle that will form the lens of the eye (lens vesicle).  The optic cup is about 550 micrometers in diameter and initially contains two layers of cells.  These cells divide quickly to form an inner neural retina and an outer retinal pigment epithelium.  The neural retina divides to form multiple layers of cells, including photoreceptor cells, which respond to light.  Austin Smith, director of the Centre for Stem Cell Research at the University of Cambridge, UK said of the developing eye: “The morphology is the truly extraordinary thing.”

Previously, stem-cell biologists were able to grow embryonic stem-cells into two-dimensional sheets, but over the past four years, Sasai and his colleagues have used mouse embryonic stem cells to grow well-organized, three-dimensional cerebral-cortex tissue (Eiraku, M., et al. Cell Stem Cell 3, 519–532, 2008)., pituitary-gland (Suga, H., et al. Nature 480, 57–62, 2011)., and optic-cup tissue (Eiraku, M., et al. Nature 472, 51–56, 2011).  His present successes represent the first time that anyone has managed a similar feat using human cells.

The fact that Sasai’s laboratory was able to grow the optic cup in the laboratory, and that it recapitulated the same developmental events in the same order shows that the cues for this the formation of the eye rely, primarily, on cue from inside the cell, rather than relying on external triggers.  “This resolves a long debate,” says Sasai, over whether the development of the optic cup is driven by internal or external cues.

This achievement could make a big difference in the clinic.  There have been increasing successes cell transplantations in the last few years.  For example, a last month, a group at University College London showed that a transplantation of stem-cell derived photoreceptors could rescue vision in mice (Pearson, R. A., et al. Nature 485, 99–103, 2012).  The transplant involved only rod-shaped receptors, not cone-shaped ones, which would leave the recipient seeing fuzzy images. Sasai’s organically-layered structure provides hope that integrated photoreceptor tissue might be transplantable someday.   The developmental process could also be adapted to treat a particular disease, and stocks of tissue could be created for transplant and frozen.

Sasai emphasized that the cells in the optic cup are differentiating and there are no embryonic stem cells in them.  This reduces concerns that transplants of these optic cups or structures derived from them might develop cancerous growths or fragments of unrelated tissues. “It’s like pulling an apple from a tree. You wouldn’t expect iron to be growing inside,” says Sasai. “You’d have no more reason to expect bone to be growing in these eyes.”

Masayo Takahashi, an ophthalmologist at the CBD, has already started transferring sheets of the retina from such optic cups into mice, and she would like to do the same with monkeys sometime this year.  The big question is whether transplanted tissue will integrate into native tissue.

The big question is whether or not clinicians and stem-cell biologists can easily repeat Sasai’s results?  Some, in fact, have already tried and failed to reproduce Sasai’s mouse experiment using human cells. “We need to know how robust, how reproducible it is,” says Smith.

The First Limbal Stem Cell Transplant with Cultured Limbal Stem Cells from a Cadaver


A genetic condition called “aniridia” results from mutations in the PAX6 gene. Approximately 1/50,000-1/1000,000 babies have aniridia. Aniridia results in the complete absence of an iris, and aniridia patients are unable to adjust to light differences.

Because mutations in the PAX6 gene are dominant, aniridia patients half a 50% chance of passing the aniridia condition to their children.

Fortunately for aniridia patients, limbal stem cells can now be cultured in the laboratory and used in clinical settings (see Di Iorio E, et al., Ocul Surf. 2010;8(3):146-53). A Scottish woman with aniridia has just received on of the first limbal stem cell transplants from a cadaver. These cadaver limbal stem cells were cultured and then transplanted onto the surface of her eye.

This woman, Sylvia Paton, who is 50 years old and from the Scottish town of Corstorphine (a west suburb of Edinburgh), is the first person in the United Kingdom to experience this ground-breaking treatment in February of 2012. Her procedure will hopefully reduce her vision problems and ready her for another procedure whereby her lens will be replaced.

For this procedure, limbal stem cells from a dead donor were cultured in the laboratory. The cells were attached to a membrane and then transplanted onto the surface of the left eye. The operation took a total of three hours.

Before her operation, Mrs. Paton could previously only see dark and light through her eye, but this treatment should repair her cornea, and prepare her for another surgery one year later to remove her cataract.

Dr Ashish Agrawal, the National Health Service consultant ophthalmologist who performed the operation, said: “It is now 12 weeks since the transplant and I am delighted to report that Sylvia is recovering well. Her cornea is clear and I hope that it will continue to maintain clarity. However, this is the first and the major step in the complex visual rehabilitation process and she will require further surgical treatment to restore vision.”

We wish Mrs. Paton well and hope that her vision continues to improve.