Update on First Induced Pluripotent Stem Cell Clinical Trial


Masayo Takahashi, an ophthalmologist at the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, has pioneered the use of induced pluripotent stem cells (iPSCs) to treat patients with degenerative retinal diseases.

Takahashi isolated skin cells from her patients, and then had them reprogrammed into iPSCs in the laboratory through a combination of genetic engineering and cell culture techniques. These iPSCs have many similarities with embryonic stem cells, including pluripotency, which is the potential to differentiate into any adult cell type.

Once induced pluripotent stem cell lines were established from her patient’s skin cells, they had their genomes sequenced for safety purposes, and then differentiated into retinal pigmented epithelial (RPE) cells. RPE cells lie beneath the neural retina and support the photoreceptors that respond to light. When the RPE cells die off, the photoreceptors also begin to die.

Takahashi watched the transplantation of the RPE cells that she had grown in the laboratory into the back of a woman’s damaged retina. This transplant would constitute the first test of the therapeutic potential of iPSCs in people. Takahashi described the transplant as “like a sacred hour.”

Takahashi has collaborated with Shinya Yamanaka, the discoverer of iPSC technology. She devised ways to convert the iPS cells into sheets of RPE cells. She then tested the resulting cells in mice and monkeys, jumped the various regulatory loops, recruited patients for her clinical trial, and practiced growing cells from those patients. Finally, she was ready to try the transplants in people with a common condition called age-related macular degeneration, in which wayward blood vessels destroy photoreceptors and vision. The transplants are meant to cover the retina, patch up the epithelial layer and support the remaining photoreceptors. Watching the procedure, “I could feel the tension of the surgeon,” Takahashi said.

This transplant surgery occurred approximately a year ago. Some new data on this patient is available.

As of 6 months after the transplant, the procedure appears to be safe. The one-year safety report should appear soon. Prior to the transplant, the patient was a series of 18 anti-vascular endothelial growth factor (anti-VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease. However, data presented by Dr. Takahashi showed that the patient had subretinal fibrotic tissue removed during the transplant surgery in order to make room for the RPE cells. Once the RPE cells were implanted, the patient experienced no recurrence of neovascularization at the 6-month point. This is significant because she has not had any other anti-VEGF injections since the transplant. Her visual acuity was stabilized and there have been no safety related concerns to date.

I must grant that this is only one patient, but so far, these results look, at least hopeful. Hopefully other patients will be treated in this trial, and hopefully, they will experience the same success that the first patient is enjoying. We also hope and pray that the first patient will continue to experience relief from her retinal degeneration.

As to the treatment of the second patient of this trial, Takahashi has hit a snag. Some mutations were detected in the iPS cell-derived RPE cells prepared for the second patient. No one knows if these mutations make these cells dangerous to implant. Regulatory guidelines, at this point, are also no help. Apparently, the cells have three single-nucleotide change and three copy-number changes that are present in the RPE cells that were not detectable in the patient’s original skin fibroblasts. The copy-number changes were, in all cases, single-gene deletions. One of the single-nucleotide changes is listed in a database of cancer somatic mutations, but only linked to a single cancer. Further evaluation of these mutations shows that they were not in “driver genes for tumor formation,” according to Dr. Takahashi.

Tumorigenicity tests in laboratory animals has established that the RPE cells are safe. Remember that the presence of a mutation does not necessarily mean that these RPE cells can be tumorigenic.

However, Takahashi has still decided to not transplant these cells into the second patients. Part of the reason is caution, but the other reason is compliance with new Japanese law on Regenerative Medicine, which became effective after iPS trial was begun. This law, however, does not specify how safe a cell line has to be before it can be transplanted into a patient.

RIKEN’s decision to halt the trial is probably a good idea. After all, this is the first trial with iPSCs and it is important to get it right. Even though the RPE cells were widely thought to be safe to use, Takahashi decided not to implant another patient with RPEs derived from their own cells. Instead, they decided to use RPEs made from donated iPSC lines. Therefore, Takahashi is in discussions government officials to determine how this change of focus for the trial affects their compliance with Japanese law.

Frankly, this might be a very savvy move on Takahashi’s part. As Peter Karagiannis, a spokesperson for the Center for iPS Cell Research and Application, noted: “As of now, autologous would not be a feasible way of providing wide-level clinical therapy. At the experimental level it’s fine, but if it’s going to be mass-produced or industrialized, it has to be allogeneic.”

Therefore, the RIKEN institute is moving forward with allogeneic iPSC-derived RPEs. RIKEN will work in collaboration with the Center for iPS Cell Research and Application (CiRA) in Kyoto, Japan, which has several well characterized, partially-matched lines whose safety profiles have been established by strict, rigorous safety testing methods. However, immunological rejection remains a concern, even if these cells are transplanted into an isolated tissue like the eye where to immune system typically is not allowed. The simple fact is that no one knows if the cells will be rejected until they are used in the trial.

An additional concern is that CiRA has not typed its cells for minor histocompatibility antigens, which can cause T cell–mediated transplant rejection.

Nevertheless, Takahashi and her team deserve a good deal of credit for their work and vigilance.

Pioneering Stem Cell Trial Halted Over Mutations in Stem Cells


A halt has been called to a pioneering stem cell clinical trial after genetic mutations were detected in the cells derived from one of the trial participants; cells that were to be used to treat the patient.

What makes this trial so unique is that it is the first to investigate if cells derived from induced pluripotent stem (iPS) cells can be used to treat disease. iPS cells are made from mature, adult cells that have been genetically engineered to transiently express four genes (Oct-4, Soc-2, Klf-4, and c-Myc), and then cultured in the laboratory in embryonic stem cell-type medium. This treatment kills many of the cells, but a fraction of them are developmentally regressed into a pluripotent stem-cell-like state. From this pluripotent state, the cells can be differentiated into almost any other type of cell in the body. Such differentiated cells can them be transplanted back into the body of the patient to replace diseased, dying cells.

In this trial, skin cells from trial participants were reprogrammed into iPS cells, which were then differentiated into retinal cells. Transplantation of these retinal cells could potentially interrupt or, perhaps even reverse, the damage caused by a disease called age-related macular degeneration, which leads to loss of vision and, potentially, blindness. The first patient in the trial, a 70-year-old woman, was treated in September, 2014, and is reportedly in good health.

Treatment of the second patient, however, has hit a snag. “A mutation was found in the cells before transplantation into the second patient, and this is something we took into account when we made the decision to suspend the study for the time being,” says trial leader Masayo Takahashi of the Riken Center for Developmental Biology in Kobe, Japan.

Analyses of the iPS cells made from skin cells taken from the second patient revealed six mutations. Three of these mutations consisted of deletion of particular genes, and the other three consisted of changes to genes, including one in an oncogene (a gene with the potential to cause cancer), although this one is linked with a low risk. The mutations were not detectable in the original skin cells, which suggests that they occurred as a result of the iPS-cell procedure. However, other work has shown that low-frequency mutations in the initial cells that are difficult to detect can become amplified in iPS cells derived from  that cell population.

“Either they were there at undetectable levels in the skin cells, or they were caused by the iPS cell induction process,” says Shinya Yamanaka of Kyoto University in Japan, one of the scientists who developed the iPS cell reprogramming technique. “However, the risk of carcinogenesis was considered low.”

Other factors that caused the trial to be halted are regulatory changes in Japan. Takahashi told the magazine New Scientist that the law now stipulates that in Japan only certain institutions can run stem-cell trials. Once the team has worked out how to accommodate these changes, they hope to resume work and test five more people using healthy, mutation-free skin cells from younger people.

“I think it’s an easily fixable problem if they go this route,” says Robert Lanza, chief scientist at Ocata Therapeutics in Marlborough, Massachusetts, which is also developing stem-cell therapies for age-related blindness.

Regardless, the discovery of mutations that could be related to the process by which iPS-cells are derived is troubling, and is a concern that stem cell scientists have had since iPS cells were first discovered. One of the benefits of stem-cell therapies is that the cells can multiply rapidly, which is also a characteristic shared by cancer cells.

But that similarity doesn’t necessarily mean cancer will develop. “It’s important to understand that even mutations in oncogenes don’t guarantee that cancer will result,” says Jeanne Loring of the Scripps Research Institute in La Jolla, California.

“It will be important to determine the source of the mutation before jumping to conclusions that reprogramming cells will always carry this sort of risk,” says Mike Cheetham of the Institute of Ophthalmology at University College London. “It will be important to determine the source of the mutation before jumping to conclusions”

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.”

Sweat Glands Are A Source of Stem Cells for Wound Healing


Stem Cells from human sweat glands serve as a remarkable source for wound healing treatments according to a laboratory in Lübeck, Germany.

Professor Charli Kruse, who serves as the head of the Fraunhofer Research Institute for Marine Biotechnology EMB, Lübeck, Germany, and his colleagues isolated cultured pancreatic cells in the course of their research to look into the function of a protein called Vigilin. When the pancreatic cells were grown in culture, they produced, in addition to other pancreatic cells, nerve and muscle cells. Thus the pancreas contains a stem cell population that can differentiate into different cell types.

Kruse and his group decided to investigate other glands contained a similar stem cell population that could differentiate into other cell types.

Kruse explained: “We worked our way outward from the internal organs until we got to the skin and the sweat glands. Again, this yielded the same result: a Petri dish full of stem cells.”

Up to this point, sweat glands have not received much attention from researchers. Mice and rats only have sweat glands on their paws, which makes them rather inaccessible. Human beings, on the other hand, have up to three million sweat glands, predominantly on the soles of out feet, palms of the hand, armpits, and forehead.

Ideally, a patient could have stem cells taken from her own body to heal an injury, wound, or burn, Getting to these endogenous stem cell populations, however, represents a challenge, since it requires bone marrow biopsies or aspirations, liposuction, or some other invasive procedure.

Sweat glands, however, are significantly easier to find, and a short inpatient visit to your dermatologist that extracts three millimeters of underarm skin could provide enough stem cells to grow in culture for treatments.

Stem cells from sweat glands have the capacity to aid wound healing. Kruse and his group used sweat gland-based stem cells in laboratory animals. The Kruse group used skin biopsies from human volunteers and separated out the sweat gland tissues under a dissecting scope. Then the sweat gland stem cells were grown in culture and induced to differentiate into a whole host of distinct cell types.

Then Kruse’s team grew these sweat gland stem cells in a skin-like substrate that were applied to wounds on the backs of laboratory animals. Those animals that had received stem cell applications healed faster than those that received no stem cells.

If the stem cells were applied to the mice with the artificial substrate, the cells moved into the bloodstream and migrated away from the site of the injury. In order to help heal the wound the cells had to integrate into the skin and participate in the healing process.

“Not only are stem cells from sweat glands easy to cultivate, they are extremely versatile, too,” said Kruse.

Kruse and his team are already in the process of testing a treatment for macular degeneration using sweat gland-based stem cells. “In the long-term, we could possibly set up a cell bank for young people to store stem cells from their own sweat glands/ They would then be available for use should the person need new cells, following an illness,l perhaps, or in the event of an accident,” Kruse said.

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.

A Protein from Fat-Based Stem Cells Prevents Light-Induced Damage to the Retina


Japanese researchers from Gifu Pharmaceutical University and Gifu University have reported that a type of protein found in stem cells taken from adipose (fat) tissue can reverse and prevent age-related, light-induced retinal damage in mice. These results may lead to treatments for patients faced with permanent vision loss.

According to the work done by these two research teams led by Drs. Hideaki Hara and Kazuhiro Tsuruma, a single injection of fat-derived stem cells (ASCs) reduced the retinal damage induced by light exposure in mice. This study also discovered that when fat-derived stem cells were grown in culture with retinal cells, the stem cells prevented the retinal cells from suffering damage after exposure to hydrogen peroxide and visible light both in the culture and in the retinas of live mice.

Additionally, Hara and Tsuruma and their colleagues discovered a protein in fat-derived stem cells called “progranulin.” This protein, progranulin, seems to play a central role in protecting other cells from suffering light-induced eye damage.

In the retina, which lies at the back of the eye, excessive light exposure causes degeneration of the photoreceptor cells that respond to light. Several studies have suggested that a long-term history of exposure to light might be an important factor in the onset of age-related macular degeneration. Photoreceptor loss is the primary cause of blindness in particular eye-specific degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

“However, there are few effective therapeutic strategies for these diseases,” Hideaki Hara, Ph.D., R.Ph., and Kazuhiro Tsuruma, Ph.D., R.Ph.

“Recent studies have demonstrated that bone marrow-derived stem cells protect against central nervous system degeneration with limited results. Just like the bone marrow stem cells, ASCs also self-renew and have the ability to change, or differentiate, as they grow. But since they come from fat, they can be obtained more easily under local anesthesia and in large quantities.”

The fat tissue used in the study was taken from mice and processed in the laboratory to isolate the fat-based stem cells. Afterwards, those cells were tested with cultured mouse retinal cells, and they show a robust protective effect. These successes suggested to the team to test their theory on a live group of mice that had retinal damage after exposure to intense levels of light.

Five days after receiving injections of the fat-based stem cells, the animals were tested for photoreceptor degeneration and retinal dysfunction. The results showed the degeneration had been significantly inhibited.

“Progranulin was identified as a major secreted protein of ASCs, which showed protective effects against retinal damage in culture and in animal tests using mice,” Drs. Hara and Tsuruma said. “As such, it may be a potential target for the treatment of degenerative diseases of the retina such as age-related macular degeneration and retinitis pigmentosa. The ASCs reduced photoreceptor degeneration without engraftment, which is concordant with the results of previous studies using bone marrow stem cells.”

“This study, suggesting that the protein progranulin may play a pivotal role in protecting against retinal light-induced damage, points to the potential for new therapeutic approaches to degenerative diseases of the retina,” said, Anthony Atala, MD, editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine, where this work was published.

Japanese first Ever Induced Pluripotent Stem Cell Clinical Trial Given the Green Light


The first clinical trial that utilizes induced pluripotent stem cells has been given a green light. For this clinical trial six patients who suffer from age-related macular degeneration will donate skin biopsies and the cells from these skin biopsies will be used to generate induced pluripotent stem (iPS) cells in the laboratory. After those iPS cell lines are screened for safety (normal numbers of chromosomes, no mutations in critical genes, etc.), they will be differentiated into retinal cells. The retinal cells will be transplanted into the retinas of these six patients.

This clinical trial was approved by Japan Health Minister Norihisa Tamura and it will be next summer by Masayo Takahashi. Dr. Takahashi is a retinal regeneration expert and a colleague of the man who first developed iPS cells, Shinya Yamanaka. Yamanaka won the Nobel Prize for his discovery of iPSCs last year. In fact, this clinical trial epitomizes, in the eyes of many, the determination of Japanese scientists and politicians to dominate the iPS cell field. This national ambition kicked into high gear after Yamanaka shared the Nobel Prize for Physiology or Medicine last October for his iPS cell work.

Norhisa Tamura, Japanese Minister of Health
Norihisa Tamura, Japanese Minister of Health
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.

“If things continue this way, this will be the first in-clinic study in iPS cell technology,” says Doug Sipp of the Riken Center for Developmental Biology (CDB). The CDB, Takahashi’s institute, will co-run the trial with Kobe’s Institute for Biomedical Research and Innovation. “It’s exciting.”

Sipp, however, also noted that this move has not surprised anyone in Japan, since the Japanese stem cell community has heavily invested in iPS cells. Nevertheless, since Takahashi yet to formally publish the details of her trial, some have questioned whether she is actually ready to move forward. IPS cells are viewed as the perfect compromise for regenerative medicine. They are adult, and therefore do not require the destruction of human embryos for their establishment, and they are also pluripotent like an embryonic cell, which makes them relatively powerful sources for regenerative medicine.

Critics, however, warn that iPS cells were only discovered in 2007. To date, they remain difficult to create and culture and they can become tumorous in many hands. However, many labs have a great deal of expertise and skill when it comes to handling and deriving iPS cells. These labs derive and culture iPS cells routinely. In fact, Sipp notes that Riken’s CDB alone has produced world-class work with all kinds of stem cells, including embryonic stem (ES) cells, which are the models for iPS cells.

Additionally, Sipp and others point out that a scientist who has collaborated with Takahashi in the past, Riken’s Yoshiki Sasai, is doing groundbreaking work with ES cells and the eye. The British journal Nature has called Sasai “The Brainmaker,” and has said that his research is “wowing” the world.

The Japanese government has also soundly funded Takahashi’s trail. The health ministry’s recent stimulus plan set aside more money for stem cells (in particular iPS cells) than anything else. According to the journal Nature, the Japanese government sequestered 21.4 billion yen ($215 million) for stem cell research. Of this pot of money, the health ministry provided 700 million yen ($7 million) for a cell-processing center to support Takahashi before her trial was even approved. Two centers devoted to iPS cells are slated to be built with 2.2 billion yen ($22 million). The AFP reports the prime minister has set aside a breathtaking $1.18 billion, for iPS-cell work. Yamanaka has told Nature that the Japanese government seems to be “telling us to rush iPS cell-related technologies to patients as quickly as possible.”

Robert Lanza, CSO of Advanced Cell Technology, might once have been the logical bet to be first to the clinic with iPS cells. Unlike Takahashi, he has three ES cell trials under his belt, and has started talks with the FDA about transplanting iPS cell-derived platelets, but his iPS proposal is taking longer. Lanza bitterly noted, not without justification, “We don’t have the prime minister and emperor to speed things along for us.”

Since 2007, the year that Yamanaka reported the derivation of iPS cells from adult cells, Japan has focused on iPS cells. Yamanaka showed that increasing the expression of four genes could change limited adult human cells into potent, embryonic-like cells. “At Yamanaka’s institute alone, there are at least 20 teams focusing on iPS cells now,” Sipp says. There are teams at Riken, the Universities of Tokyo and Keio, and others. “A lot is happening here.” In fact, the Center for IPS Cell Research and Application was created expressly for Yamanaka.

Takahashi has reported part of the design of her clinical trial at scientific meetings. She told the International Society for Stem Cell Research in June 2012 she had created iPS-cell derived retinal pigment epithelial (RPE) cells for transplantation. RPE cells lie behind the photoreceptors in the retina, and the photoreceptors have their ends embedded into the RPE. The RPE cells replenish and nourish the photoreceptors, and without the RPE cells, the photoreceptors die from the damage incurred by exposure to light.

Retinal Pigmented Epithelium

Death of the RPE cells cause eventual death of photoreceptors and that results in blindness. At the International Society for Stem Cell Research conference, Takahashi reported her that her iPS cell-derived RPEs possess proper structure and gene expression. They also do not produce tumors when transplanted into mice, and survive at least six months when transplanted into the retinas of monkeys. The vision of these animals, however, was not tested. She did note that some AMD patients’ sight improves when RPE cells are moved from the eye’s periphery to its center.

Retinal pigment epithelial cells derived from iPS cells.
Retinal pigment epithelial cells derived from iPS cells.

Takahashi has published many iPS and ES cell papers. These papers include two papers with Yamanaka: one on creating retinal cells from iPS cells, and one on creating safe iPS cells. However she has not published trial details, which is not required, but such a landmark trial should be transparent, as argued by many stem cell experts.

Still, according to Sipp, Takahashi has submitted a relevant paper to a top journal for review, which shows that this clinical trial is purely a determination of the safety of the procedure. Lanza has reported his trials in the journal The Lancet, and similar, but small, trials are doing well. His three ES cell trials treated Stargardt’s macular dystrophy and Age-related Macular Degeneration. Lanza’s trial, however, treated “dry” macular degeneration, while Takahashi’s trial will treat “wet” Age-related Macular Degeneration, which is good news for Takahashi.

Paul Knoepfler, a UC Davis stem cell scientist who runs a widely read blog site, has written that the ministry overseeing Takahashi’s trial will reportedly monitor some key factors: gene sequencing and tumorigenicity. But Knoepfler, like others, would like to see more details.

The Japanese Health Ministry and the US FDA recently agreed to devise a joint regulatory framework for retinal iPS cell clinical trials, which will come on line 2015. Takahashi’s trial is set for 2014.