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.

Stem Cells Preserve Vision in an AMD-Like Model


Stem cell transplantation is a promising potential treatment for retinal degenerative diseases. Because retinal degeneration often leads to blindness, stem cells might be one of the up-and-coming tools in the battle against blindness.

The laboratory of Shaomei Wang (Cedars-Sinai Medical Center, Los Angeles) have assessed the effectiveness of stem cell-based therapeutic strategies using the Royal College of Surgeons (RCS) rat model, which mimics the disease progression of age-related macular degeneration (AMD). In RCS mice, the retinal pigment epithelium or RPE degenerates and is disrupted, which leads to the death of photoreceptors (Mullen RJ and LaVail MM Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 1976;192:799-801). The work by Wang and his colleagues has shown that human cortical-derived neural progenitor cells (hNPCctx) could dramatically rescue vision in the RCS rat (see Wang S, and others, Investigative ophthalmology & visual science 2008;49:3201-3206; Gamm DM, and others, Wang S, Lu B, et al. PLoS One 2007;2:e338). Unfortunately, the fetal origin of these cells presents an obstacle, because such cells are not readily available and come from aborted fetuses.

To overcome such obstacles, Wang and his colleagues assessed the ability of a stable neural progenitor cell line (iNPCs) derived from induced pluripotent stem cells (iPSCs) to preserve vision after sub-retinal injection into RCS rats (Sareen D, and others, J Comp Neurol 2014;522:2707-2728). A report in the journal Stem Cells by Wang and others establishes that iNPC injection leads to the reversal of AMD-related symptoms, the preservation of visual function, and may represent a patient-specific therapeutic option (Tsai Y, and others, Stem Cells 2015;33:2537-2549).

Wang and his others showed that an iNSC-treated eye scored higher in all functional tests used (optokinetic response (OKR), electroretinography (ERG), and luminance threshold responses (LTR)), compared to an untreated eye, in RCS mice at 150 days post-transplant. This improvement nicely correlates with the improved protection of photoreceptors in iNPC-treated eyes, which presented with normal cone morphology and the reversal of disease-associated changes throughout the retina.

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So how do iNPCs help preserve the photoreceptors and visual function? Wang and his team found that iNPCs survived up to 130 days in RCS retinas, which when normalized to lifespan, represents around 16 years in humans. Additionally, they discovered that iNPCs were able to migrate to an area between the retinal pigment epithelial and photoreceptor layers. This allows the injection of cells into non-affected neighboring regions of the retina, which will not to worsen any compromised retinal components. iNPCs did, however, continue to express NSC/NPC markers and did not mature neural/retinal markers, suggesting that grafted-iNPCs remained phenotypically uncommitted progenitor cells and did not differentiate towards a retinal phenotype.

Further investigations found that iNPC-treatment reduced levels of toxic undigested bits of the photoreceptor cell membranes. Accumulation of these photoreceptor outer segments (POS) cause the photoreceptors to die off. Typically, the RPE cells goggle up these toxic membrane bits, degrade them, and recycle their components for the photoreceptors. The fact that these POS bits were not accumulating in the retinas of RCS mice suggested to Wang and his colleagues that the grafted-iNPCs restored POS degradation in RCS rats. They subsequently found that iNPCs expressed phagocytosis-related genes and could gobble up and degrade POS in culture. They extended these findings in living creatures by identifying the different stages of POS digestion and even viewed engulfed membranous discs inside the cytoplasm of iNPCs.

Overall, iNPC injection appears to be a safe and effective long-term treatment for Acute Macular Degeneration in the RCS rat preclinical model, and holds great promise for the translation into a patient-specific treatment for the preservation of existing retinal structure and vision during the early stages of AMD in humans. Wang noted that iNPC treatment in this model occurred at later stages of degeneration, which represents a more clinical relevant stage. However, an unstudied possibility is restoring phagocytosis by iNPCs to treat loss of visual acuity early on in the course of the disease.

Differential Immunogenicity of Cells Derived from Induced Pluripotent Stem Cells


Induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific pluripotent stem cells may become a renewable source of a patient’s own cells for regenerative therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood.

Using a humanized mouse model (denoted Hu-mice) with a functional human immune system, Yang Xu and his colleagues from UC San Diego has shown that most teratomas or tumors formed by human iPSCs were readily recognized by immune cells and rejected. However, when these human iPSCs were differentiated into smooth muscle cells or retinal pigmented epithelial cells, the results were rather different. Human iPSC-derived smooth muscle cells appear to be highly immunogenic, but human iPSC-derived retinal pigment epithelial (RPE) cells are tolerated by the immune system, even when transplanted outside the eye.

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When Xu and others examined these results more closely, they discovered that this differential immunogenicity is due to the abnormal expression of cell surface proteins in hiPSC-derived Smooth Muscle Cells, but not in hiPSC-derived RPEs.

These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration. They also show that iPSC lines must be individually screened to determine if their derivatives are recognized by the patient’s immune system as foreign.

These results were published in Cell Stem Cell.

Embryonic Stem Cell-Derived Retinal Cells Treat Blindness in Eye Patients


Embryonic stem cells are derived from human embryos, can only grow in culture indefinitely, and have the ability to potentially differentiate into any adult cell type in the human body.  Because cell and tissues made from embryonic stem cells bear the same tissue types as the embryos from which they were derived, they will be rejected by the immune system patient.  However, there are sites in our bodies were the immune system does not go, and that includes the central nervous system and the eyes.  This is the reason why clinical trials with embryonic stem cell-derived cells have focused, to date, on spinal cord injuries and eye diseases.

Several clinical trials have examined the ability of retinal pigmented epithelial (RPE) cells made from embryonic stem cells to treat patients with dry macular degeneration or an inherited eye disease called Stargardt’s disease.  Data from these trials has been reported in an article in the medical journal The Lancet, and accordingly, none of the treated patients showed tumor formation or immunological rejection of the implants and, most impressively perhaps, partial blindness was reversed in about half of the eyes that received transplants.

The results might re-energize the quest to harness embryonic stem cells for human medicine.  Dr. Anthony Atala of the Wake Forest Institute for Regenerative Medicine called the work “a major accomplishment” in an accompanying commentary on the article.

RPE cells lie just behind the photoreceptor cells in the retina of our eyes.  Photoreceptors have their ends hurried in the RPE layer.  This arrangement exists for a very good reason; the photoreceptors are exposed to high intensities of light and they suffer respectable amounts of oxidative damage.  The components of the photoreceptors cells are made in the very lowest parts of the RPEs and then are eventually pushed to the ends of the cells.  At the end of the photoreceptor cells, the RPEs relieve the photoreceptors of their photodamaged parts and gobble them down, and recycle the cellular components.  Thus, RPE cells serve a photoreceptor cell repair and service cells.  If the RPE cells begin to die, the photoreceptors are not long the this work either.

In the case of dry macular degeneration, which accounts for 90 percent of diagnosed cases of macular degeneration, the light-sensitive photoreceptor cells of the macula (the portion of the retina were the day vision is the sharpest) slowly break down. Damage to the macula causes blurring or spotty loss of central vision and yellowish cellular deposits called drusen (extracellular waste products from metabolism) form under the retina between the retinal pigmented epithelium (RPE) layer and a basement membrane called Bruch’s membrane, that supports the retina. An increase in the size and number of drusen is associated with the death of RPE and, consequently, photoreceptor cells, and is sometimes the first sign of dry macular degeneration.

Medical illustration of dry macular degeneration

Mutations in several genes have been identified in families with dry macular degeneration that increase the risk for dry macular degeneration.  These include the SERPING1 gene, those genes that encode the complement system proteins  factor H (CFH), factor B (CFB) and factor 3, and fibulin-5.  Additionally, some environmental and behavioral factors also influence the risk a person will develop macular degeneration.  These include smoking, exposure to blue light, ingestion of a high-fat diet, elevated blood pressure and serum cholesterol levels, and low vitamin D levels.

Stargardt’s disease is an inherited, juvenile form of macular degeneration that is caused by mutations in the ABCR gene.  The protein encoded by this gene is a waste metabolite transporter, and defects in this protein cause the build up of a toxic metabolite called lipofuscin in the RPE cells, which leads to their demise and the death of the photoreceptors.

In this study, the main goal was to assess the safety of the transplanted cells. The study “provides the first evidence, in humans with any disease, of the long-term safety and possible biologic activity” of cells derived from embryos, said co-author Dr. Robert Lanza, chief scientific officer of Advanced Cell Technology, which produced the cells and funded the study.

Nine patients with Stargardt’s disease and nine with dry age-related macular degeneration received implants of the retinal cells in one eye. The other eye served as a control.  Four eyes developed cataracts and two became inflamed, probably due to the patients’ age (median: 77) or the use of immune-supressing transplant drugs.

The implanted RPE cells survived in all 18 patients, most of whose vision improved.  In those with macular degeneration, treated eyes saw a median of 14 additional letters on a standard eye chart a year after receiving the cells, with one patient gaining 19 letters. The untreated eyes got worse, overall. The Stargardt’s patients had similar results.

In real-life terms, patients who couldn’t see objects under 12 feet (4 meters) tall can now see normal-size adults.

The vision of one 75-year old rancher who was blind in the treated eye (20/400) improved to 20/40, enough to ride horses again, Lanza said.  Others became able to use computers, read watches, go to the mall or travel to the airport alone for the first time in years.

While calling the results “encouraging,” stem cell expert Dusko Ilic of Kings College London, who was not involved in the work, warned that even if the larger clinical trial planned for later this year is also successful, “it will take years before the treatment becomes available.”

Other cell types can also form RPE cells and these include induced pluripotent stem cells, mesenchymal stem cells from fat (Ophthalmic Res. 2012;48 Suppl 1:1-5), adult retinal stem cells (Pigment Cell Melanoma Res. 2011 Feb;24(1):233-40), and iris pigmented epithelial cells (Prog Retin Eye Res. 2007 May;26(3):302-21).  We do not need to destroy embryos to treat eye diseases with 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.”

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.

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.