Induced Pluripotent Stem Cells Form Layered Retina-Like Structure in Culture

Embryonic stem cells can form several different types of eye-specific cells. In the early years of the 21st century, reproducible and efficient methods for differentiating embryonic stem cells into lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells were developed (Haruta M., Embryonic stem cells: potential source for ocular repair. Semin Ophthalmol. 2005 Jan-Mar;20(1):17-23).

Other experiments showed that embryonic stem cells could be differentiated into neural progenitor cells (NPCs). These NPCs differentiated in culture and some of them even expressed genes characteristic of developing retinal cells. Although it must be noted that this was uncommon and cells expressing markers of mature photoreceptors were not observed. Implantation of these differentiated NPCs into the retinas of laboratory animals allowed them to survive for at least 16 weeks, migrate over large distances, and form photoreceptor-like cells that made blue-absorbing pigments. These cells also integrated into the host retina (Banin E, Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells. Steem Cells. 2006 Feb;24(2):246-57).

These early experiments were followed by several others that showed equally remarkable promise. Workers in Takahashi’s laboratory in Kobe, Japan found that embryonic stem cells could form retinal precursors, but that they rarely formed photoreceptors unless they were treated with extracts from embryonic retinas. However in a follow-up paper in 2008, Takahashi, research group found that specific cocktails of small molecules and/or growth factors could push retinal precursors to form photoreceptors (Osakada, et al., Nat Biotechnol. 2008 Feb;26(2):215-24). Kunisada’s lab in Gifu, Japan used various techniques to differentiate embryonic stem cells in culture so that they would form an elaborate retinal-like structure. When this structure was transplanted into the eyes of rodents with inherited eye diseases, these transplanted cells regenerated the ganglion cells in the retina (Aoki H, et al., Graefes Arch Clin Exp Ophthalmol. 2008 Feb;246(2):255-65). Yu’s lab from Seoul National University, Seoul, South Korea made pure RPE cell cultures from embryonic stem cells and then transplanted them into the eyes of rodents with RPE-based retinal degeneration diseases (Park UC, et al., Clin Exp Reprod Med. 2011 Dec;38(4):216-21). The transplanted cells formed RPEs and integrated into the retinas of the laboratory animals. Sophisticated functional assays definitively showed that the RPEs made from embryonic stem cells gobbled up the old segments from photoreceptors and recycled the components back to the photoreceptors (Carr AJ, et al., Mol Vis. 2009;15:283-95).

Using embryonic stem cells to make retina-like structures in culture can provide a model for testing new drugs and procedures to treat degenerative eye diseased such a macular degeneration. Also, such structures might be used to transplant sections of retina into the eyes of individuals where the retina has died off.

With this goal in mind, researchers at the University of Wisconsin-Madison have succeeded in making made early retina structures that contain growing neuroretinal progenitor cells. The novelty in this experiment is that they did it using induced pluripotent stem (iPS) cells that were derived from human blood cells.

In 2011, the laboratory, of David Gamm lab, pediatric ophthalmologist and senior author of the study whose lab is at the Waisman Center, created structures from the most primitive stage of retinal development using embryonic stem cells and iPS cells derived from human skin. These structures generated the major types of retinal cells, including photoreceptors, they did not possess the layered structure found in more mature retina. Clearly something was missing t form a retinal-like structure.

The iPS cells used in this study were made by scientists at a biotechnology company called Cellular Dynamics International (CDI) of Madison, Wisconsin. CDI pioneered the technique to convert blood cells into iPS cells, and they extracted a type of blood cell called a T-lymphocyte from donor samples. These T-lymphocytes were reprogrammed into iPS cells (full disclosure: CDI was founded by UW-Madison stem cell pioneer James Thomson).

With these iPS cells, Gamm and postdoctoral researcher and lead author Joseph Phillips, used their previously-established protocol to grow retina-like tissue from iPS cells. However, this time, about 16% of the initial retinal structures developed distinct layers, which is the structure observed in a mature retina. The outermost layer primarily contained photoreceptors, whereas the middle and inner layers harbored intermediary retinal neurons and ganglion cells, respectively. This particular arrangement of cells is reminiscent of what is found in the back of the eye.

At 72 days, stem cells derived from human blood formed an early retina structure, with specialized cells resembling photoreceptors (red) and ganglion cells (green) located within the outer and inner layers, respectively. Nuclei of cells within the middle layer are shown in blue. These layers are similar to those present during normal human eye development.

These retinal structures also showed proper connections that could allow the cells to communicate information. In the retina, light-sensitive photoreceptor cells along the back wall of the eye produce impulses that are ultimately transmitted through the optic nerve and then to the brain, and this allows. Because these layered retinal structures not only had the proper cell types, but also the proper connections, these findings suggest that it is possible to assemble human retinal cells into the rather complex retinal tissues found in an adult retina. This is extremely stupefying when one considers that these structures all started from a single blood sample.

There are several applications to which these structures might be subjected. They could be used to test drugs and study degenerative diseases of the retina such as retinitis pigmentosa (a major cause of blindness in children and young adults). Also, it might be possible one day to replace multiple layers of the retina in order to help patients with more widespread retinal damage.

Gamm said, “We don’t know how far this technology will take us, but the fact that we are able to grow a rudimentary retina structure from a patient’s blood cells is encouraging, not only because it confirms our earlier work using human skin cells, but also because blood as a starting source is convenient to obtain. This is a solid step forward.” He also added, “We were fortunate that CDI shared an interest in our work. Combining our lab’s expertise with that of CDI was critical to the success of this study.”

This work was published in the March 12, 2012 online issue of Investigative Ophthalmology & Visual Science. The research is supported by the Foundation Fighting Blindness, the National Institutes of Health, the Retina Research Foundation, the UW Institute for Clinical and Translational Research, the UW Eye Research Institute and the E. Matilda Ziegler Foundation for the Blind, Inc.

Bone Marrow Stem Cells Make the Blind (Lab Animals) See

There has been a great deal of discussion of embryonic stem cell-derived retinal pigment cells and the transplantation of these cells into the retinas of two human patients who subsequently showed improvements in their vision. One of these patients had a degenerative eye disease called “Stargardt’s macular dystrophy,” and the other had dry, age-related macular degeneration.

Stargardt Macular Dystrophy (SMD) is one of the main causes of eyesight loss in younger patients (affects 1/10,000 children), and retinal damage begins somewhere between the ages of 6 – 20. Visual impairment is usually not obvious to the patient until ages 30 – 40. Children with SMD usually notice that they have difficulty reading. They may also complain that they see gray, black or hazy spots in the center of their vision. Additionally, SDM patients take a longer time to adjust between light and dark environments.

Mutations in the ABCA4 gene seem to be responsible for most cases of SDM.  Defects in ABCA4 prevent the photoreceptors from disposing of toxic waste products that accumulate within build up in the disc space of the photoreceptors.  These toxic waste products are a consequence of housing light-absorbing pigments, and intense light exposure.  The pigment, all-trans retinal, binds to membrane lipids, and this forms a compound called NRPE (short for N retinylidene-phosphatidylethanoliamne, which is a mouth-full).  The protein encoded by ABCA4 moves NRPE into the cytoplasm of the photoreceptor cells, but if ABCA4 is not functional, NRPE accumulates in the disc space and binds more all-trans retinal to form a toxic sludge called “lipofuscin.”  Lipofuscin is taken up from the photoreceptors by the RPE cells and it kills them (see Koenekoop RK. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthal Genet. 2003;24(2):75–80).  Mutations in other genes (ELOVl4, PROM1, and CNGB3) also cause SDM.

Dry, age-related macular degeneration is associated with the formation of small yellow deposits in the retina known as “drusen.”  Drusen formation leads to a thinning and drying of the macula that eventually causes the macula to lose its function.  There is loss of central vision and the amount of vision loss is directly related to the amount of drusen that forms.  Early stages of age-related macular degeneration is associated with minimal visual impairment, but is characterized by large drusen and abnormalities in the macula.  Drusen accumulates near the basement membrane of the retinal pigment epithelium.  Almost everyone over the age of 50 has at least one small druse deposit in one or both eyes.  Only those eyes with large drusen deposits are at risk for late age-related macular degeneration.

All of this is to say that these diseases are progressive.  They have no cure and little can be done for treatment.  Secondly, people rarely get better.  However, both patients in this study showed quantifiable improvements.  The patient with age-related macular degeneration went from being able to see 21 letters in the visual acuity chart (20/500 vision for the patient, with 20/20 being perfect vision) to 28 letters (20/320).  This improvement remained stable after 6 weeks.  The patient with SMD was able to detect hand motions only, but after the stem cell injection, she could count fingers and see one letter in the eye chart by week 2, and was able to see five letters (20/800) after 4 weeks.  She also was able to see colors and contrast better and had better dark adaptation in the treated eye.

Now there are some caveats for this report.  First of all, the patient with SMD showed distinct structural improvements in the retina of the injected eye.  This patient also had distinct improvements in visual acuity.  However, the patient with dry, age-related macular dystrophy had no detectable structural improvements in the injected eye. The paper states, “Despite the lack of anatomical evidence, the patient with macular degeneration had functional improvements.”  Additionally, the non-injected eye also showed some visual improvements.  Note the words of the paper:  “Confounding these apparent functional gains in the study eye, we also detected mild visual function increases in the fellow eye of the patient with age-related macular degeneration during the postoperative period.”  Therefore, this experiment is highly preliminary and has equivocal results.  The SMD patient does show recognizable improvements, but this is only one patient.

While we are considering the efficacy of embryonic stem cells in the treatment of retinal degenerative diseases, a paper that was published in 2009 shows that bone marrow stem cells that have a cell surface marker celled “CD133” can become retinal pigment (RPE) cells.  This paper was published in the journal “Stem Cells,” and the principal author was Jeffrey Harris who did his work in the laboratory of Edward W. Scott at the University of Florida.  These cells were extracted from the bone marrow of mice and implanted into the retinas of albino mice.  Since the donor mice had pigmented skin and fur coats, the bone marrow cells were capable of making pigmented cells.  Once the CD133 cells were implanted, they survived and became pigmented.  When examined in postmortem sections, it was exceedingly clear that the transplanted CD133 cells expressed RPE-specific genes and assumed a RPE-like morphology.  Additionally, the implanted bone marrow cells also contributed functional recovery of retina.  A second set of experiments showed that human CD133 cells from umbilical cord could also integrate into mouse retinas and differentiate into RPEs.

This paper shows that embryonic stem cells are probably not necessary for retinal repair of RPE-based retinal degeneration.  Umbilical cord CD133 stem cells or bone marrow stem cells can differentiate into RPEs when transplanted into the retina.  While this paper does not address whether or not such differentiation occurs in human patients, such results definitely warrant Phase I studies. Thus once again, embryonic stem cells seem not be necessary.

StemCells, Inc. Announces Positive Preclinical Data for Its Human Neural Stem Cells Ability to Preserve Vision

A research team led by Raymond Lund, Ph.D., Professor Emeritus of Ophthalmology, and Trevor McGill, Ph.D., Research Assistant Professor at the Casey Eye Institute, Oregon Health and Science University have made a remarkable discovery using proprietary stem cells from StemCells, Inc. These results await publication in the European Journal of Neuroscience, and constitute positive preclinical data for StemCells, Inc. proprietary Human Central Nervous System Stem Cells (HuCNS-SC).

For these experiments, the team used Royal College of Surgeons (RSC) rats. RCS rats have an inherited form of retinal degeneration. Although the genetic defect that causes retinal regeneration was not known for many years, it was identified in the year 2000 to be due to a mutation in the Merkt gene. Mutations in the Merkt gene prevent the retinal pigment epithelium cells from scooping up outer segments of photoreceptor cells. As photoreceptor cells respond to light, their outer membrane proteins suffer photo-oxidation. Retinal pigment epithelial cells phagocytose these defective photoreceptor outer membrane segments and recycle them, which maintains photoreceptor health and function. When retinal pigment epithelium cells are unable to phagocytose photoreceptor out membrane segments, the photoreceptors accumulate photo-damage and eventually die.

To test the efficacy of HuCNS-SCs in preserving photoreceptor health, Lund and his colleagues injected HuCNS-SCs into the subretinal space of 21-day old RCS rats. They found that photoreceptors, the key cells of the eye involved in vision were protected from degeneration. Additionally, the density of healthy cone photoreceptors (those photoreceptors that help in color perception) remained relatively constant over several months. Visual acuity and luminance sensitivity tests in the injected RCS rats further corroborated the results of observed in the retinas. Apparently, the donor cells remained immature and did not differentiate throughout the seven-month experiment. However, the transplanted HuCNS-SCs underwent very little proliferation, and produced no tumors or abnormal growths. The ability of these transplanted cells to protect photoreceptors and preserve vision when injected into the retinas of RCS rats is important to human disorders of vision loss such as dry age-related macular degeneration (AMD).

Lund excitedly noted: “These results are the most robust shown to date in this animal model. One of the more striking findings is that the effect on vision was long-lasting and correlated with the survival of HuCNS-SC cells more than seven months after transplantation, which is substantially longer than other cell types transplanted into this same model. Also important, particularly for potential clinical application was that the cells spread from the site of initial application to cover more of the retina over time. These data suggest that HuCNS-SC cells appear to be a well-suited candidate for cell therapy in retinal degenerative conditions.”

Another investigator in this study, Alexandra Capela, Ph.D., a senior scientist at StemCells, commented, “This study showed that the HuCNS-SC cells persisted and migrated throughout the retina, with no evidence of abnormal cell formation, which supports our hypothesis of a single transplant therapeutic. With this research, then, we have shown that vision can be positively impacted with a simple approach that does not require replacing photoreceptors or the RPE cells. We look forward to investigating this promising approach in the clinic later this year.”

Phase I Study of Embryonic Stem Cell-Derived Retinal Pigment Epithelium Cells Shows Early Signs of Success

Several different diseases cause deterioration of the eye and plunge aging or even young men and women into a life of blindness. Several of these genetic diseases affect the tissues that reside at the back of the eye, which is collectively called the retina. The retina contains two main layers; an inner neural retina and an outer pigmented retina.

The neural retina is filled with photoreceptors and cells that process the outputs from the photoreceptor cells and send them to the brain. The pigmental retina contains the retinal pigmented epithelium, which plays a central role in retinal physiology. The retinal pigmented epithelium or RPE forms the outer blood-retinal barrier and supports the function of the photoreceptors. Many diseases the adversely affect the retina called “retinopathies” involve a disruption of the epithelium’s interactions with the neural retina. Other types of retinopathies are caused by uncontrolled proliferation of the RPE cells.

Transplantation of RPE cells can help treat patients that have various types of retinopathies (see Lund RD et al.,Cloning Stem Cells.2006 Fall;8(3):189-99).  However, embryonic stem cells can be made into copious quantities of RPEs rather easily (Huang Y, Enzmann V, Ildstad ST. Stem Cell Rev. 2011 Jun;7(2):434-45).  Therefore, it was only a matter of time before clinical trials were instigated with embryonic stem cell-derived RPEs.

In recent edition of the journal The Lancet, Steven Schwartz and colleagues have reported the first clinical results from patients treated with embryonic stem cell-derived RPEs.  A patient with “Stargardt’s macular dystrophy,” which is the most common form of pediatric macular degeneration, and a patient with dry age-related macular degeneration, the leading cause of blindness in the developed world, each received a subretinal injection of RPEs derived from embryonic stem cells (ESCs).  Both of these disorders are not treatable at present, but both also result from degeneration of the RPE.  Loss of RPE cells causes photoreceptor loss and progressive vision deficiency.

Schwartz and colleagues differentiated the hESCs into RPE cultures that showed greater than 99% purity.  Then they injected 50,000 RPE cells into the subretinal space of one eye in each patient. Each patient received anti-rejection drugs (low-dose tacrolimus and mycophenolate mofetil) just in case the immune system tried to attack the transplanted RPE cells.

There results are hopeful, since, after 4 months, both patients show no sign of retinal detachment, hyperproliferation, abnormal growths, intraocular inflammation, or teratoma formation.  Anatomical evidence of the injected cells was difficult to confirm in the patient with age-related macular degeneration, but was present in the patient with Stargardt’s macular dystrophy.

Both patients showed some visual improvements.  The patient who suffers from age-related macular degeneration improved in visual acuity, since she was able to recognize 28 letters in a visual acuity chart, whereas before he procedure, she was able to identify only 28 (improvement from 20/500 vision to 20/320).  The patient with Stargardt’s macular dystrophy went from counting fingers and seeing only one letter in the eye chart by week 2, and to a stable level of five letters (20/800) after 4 weeks.  This patient also showed subjective improvement in color vision, contrast, and dark adaptation in the treated eye.

These results are highly preliminary and the improvements are slight, but the progressive nature of these eye diseases suggests that the injections largely worked.  Before we can crack our knuckles for joy, we will need to see improvements with more than two patients.  But the fact that the treated eye showed improvements not seen in the untreated eye is highly suggestive that the transplanted RPEs are improving the health of the photoreceptors in the neural retina.  The eye is an ideal place to do such research because it is one place in the body that is not regularly patrolled by the immune system, and foreign cells placed in the eye tend to receive far less scrutiny from the immune system than other parts of the body.

I am glad for these patients, but I am troubled by this experiment.  Other types of stem cells can be converted into RPEs (Uygun BE, Sharma N, Yarmush M. Crit Rev Biomed Eng. 2009;37(4-5):355-75.).  Also, there are other stem cells in the eye that, if properly investigated might possess the ability to form RPEs (Bhatia B, et al.,Exp Eye Res. 2011 Dec;93(6):852-61).  Why was this experiment first done with cells that require the death of early human embryos?  The safety concerns with ESCs makes the clinical trial far more expensive and slower.  While the embryos sacrificed to make these RPEs have long since died, the ESC culture is doing some clinical good.  However, how would we feel about cell lines made from children who were murdered by a sadistic scientist?  Would you receive treatments from them given what you know about their origin?  So while this experiment shows hope, it also leads to controversy as well that is not being discussed as deeply as it should.