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.

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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.