Clinical Updates — Clinical Updates

  • Sachin Mehta, MD

So Doc, What About Stem Cells? The Potential of Stem Cells in Treating Age-Related Macular Degeneration

As vitreoretinal specialists, we've all been there. You walk into your exam room ready to see your patient with central vision loss due to atrophic age-related macular degeneration (AMD). She greets you with a stack of newspaper clippings in her lap, ready to educate you about the miracle of stem cells. You draw a deep breath, take the clippings from her hand, and start reading through them.

"So Doc," she says. "What about stem cells? I've heard they are doing some amazing things in Germany and England. Surely this must be available in the United States."

As you sit glancing over the articles while your patient discusses the television interview she saw of an elated stem cell recipient, you start to wonder to yourself, what do I really know about stem cells for the treatment of AMD? There haven't been any clinical trials in the US, and the technology is so new that it hasn't become a standard part of residency and fellowship education.

So what can I tell this patient who is so eager to find out if stem cells can restore her sight?

What are stem cells?

By definition, stem cells must be:

  1. Undifferentiated
  2. Able to differentiate into multiple cell types
  3. Capable of self-renewal through cell division
'Through the secretion of various growth and protective factors, [stem cells] have been shown to rescue diseased cells in animal models and enable return of normal cellular function.'

Stem cells represent an unlimited supply of healthy cells capable of replacing diseased or dead human tissues, such as heart, brain, liver, etc. Aside from their regenerative effects, stem cells can rescue diseased cells via their paracrine effects.[1] Through the secretion of various growth and protective factors, they have been shown to rescue diseased cells in animal models and enable return of normal cellular function.

Although stem cells have been recognized since the early 20th century, the first human embryonic stem cell (hESC) line was not established until 1998 by James Thomson at the University of Wisconsin.[2] Since then, blastocysts used for hESC research typically have come from in vitro fertilization (IVF) procedures.

In 2001, due to ethical concerns regarding the destruction of human embryos for research purposes, President George W. Bush decided to allow federal funding of research only on existing hESC lines. This ban was lifted by President Barack Obama in March 2009, but in August 2010 a US district court issued an injunction that once again froze federal funding of new hESC lines. Thus, new lines can presently only be privately funded.

What are the types of stem cells?

There are 3 broad categories of stem cells:

  1. Embryonic (ESC)
  2. Induced pluripotent (iPSC)
  3. Adult (ASC)

ESC lines are created by 1 of 2 methods:

  • Extracting blastocyst cells from IVF procedures and culturing them in a Petri dish, or
  • Extracting the nucleus of a somatic cell, such as a keratinocyte or fibroblast, injecting the nucleus into an unfertilized oocyte that has had its nucleus removed, allowing the egg to grow in culture medium into a blastocyst, and finally extracting the blastocyst cells and culturing them in a Petri dish.

With the second method, IVF is unnecessary and therefore scientists are unencumbered by the scarcity of IVF procedures dedicated for stem cell research.

The creation of iPSCs is relatively new; this process was first described by Takahashi and Yamanaka in 2006.[3] iPSCs are cells that possess ESC qualities, including the ability to differentiate into multiple cell types and to replicate in culture. They are created by exposing adult somatic cells such as fibroblasts to 4 specific transcription factors via viral vectors (ie, gene therapy), direct delivery of reprogramming proteins,[4] or small molecules.[5]

Creating iPSCs was a major achievement in stem cell research, primarily for 2 reasons:

  • They appear to have the same pluripotent potential of ESCs, but iPSCs bypass ethical concerns over destroying human embryos.
  • Immune rejection after transplantation is not a concern because the cells are autologous, whereas ESCs are not. This is important because the immune privilege of the subretinal space is likely compromised when the RPE is damaged in neurodegenerative retinal disease.[1]

ASCs are present in adult tissues and serve mainly a local tissue repair role. For example, retinal progenitor cells capable of differentiating into photoreceptors, retinal pigment epithelial (RPE) cells, bipolar cells, and Müller cells are found at the margin of the ciliary body.

These cells would theoretically be ideal retinal precursors for transplantation since they are derived from an autologous source. However, the scarce supply and the challenge to access these cells make them unlikely candidates to replace ESCs and iPSCs.

How can stem cells be used to treat AMD?

AMD is the most common cause of blindness among the elderly in the developed world and affects more than 30 million people worldwide. The recent development of anti-VEGF agents for treatment of exudative AMD has given hope of stabilization and even visual improvement to millions of people. Unfortunately, the effect of these agents is transient, and injections are required indefinitely.

There is no known treatment for nonexudative AMD, but evidence suggests that antioxidants may decrease the risk of progression to advanced forms of AMD.[6] For patients who have suffered central RPE atrophy without significant subretinal scarring, procedures such as allogeneic RPE transplantation,[7] [8] autologous RPE transplantation,[9] and macular translocation[10] have met with little success.

'AMD and other neuroretinal degenerative diseases appear to be ideal candidates for stem cell therapy...'

Transplantation of allogeneic RPE cells almost invariably leads to graft rejection without systemic immunosuppression. The other 2 procedures can be challenging to perform and have been associated with complications such as retinal hemorrhage and detachment. Also, the "new" submacular RPE cells have the same genetic makeup as the old degenerated cells, so their fate will likely be the same.[11]

By adding specific growth and differentiation factors to the culture medium, scientists can induce stem cells to transform into almost any desired cell type, including RPE cells and photoreceptors.[12] In murine and rat models of retinal degeneration, subretinal injection of ESC-[13-15] and iPSC[16] [17]-derived RPE cells have led to stable integration into areas of RPE atrophy as well as temporary rescue of retinal degeneration and preservation of visual function.

In one study, the cells survived implantation and rescued visual function in a rat model of retinal degeneration for more than 8 months.[18] Recent studies suggest that ESC-derived RPE cells are more akin to in vivo RPE cells in morphology, gene expression, immunohistochemistry, and function than existing human RPE cell lines.[12]

Why is retinal disease well-suited to stem cell therapy?

AMD and other neuroretinal degenerative diseases appear to be ideal candidates for stem cell therapy for a number of reasons:

  1. Direct monitoring of the therapeutic response is easily achieved by slit-lamp examination, fundus photography, fluorescein angiography, and other diagnostic tests.
  2. RPE cells can be easily differentiated from stem cells; thus, it is less likely that pluripotent stem cells with tumor-inducing capabilities would be accidentally transplanted into the subretinal space.
  3. AMD manifests later in life, so rapid retinal degeneration after transplantation of autologous iPSC grafts would be highly unlikely, even though the same genetic mutations would probably be present.
  4. Stem cell-derived RPE cells can be precisely delivered to their target location of RPE atrophy under direct visualization with an operating microscope.[1]

Unfortunately, RPE transplantation alone is likely not enough to restore visual function in the stem cell-mediated treatment of AMD. Photoreceptor dysfunction and atrophy are believed to be a secondary phenomenon caused by degeneration of the underlying RPE. Stem cell-derived photoreceptor transplantation has proven to be much more challenging than RPE transplantation because it requires neural integration and synapse formation.

There is evidence in animal models that transplanted photoreceptor precursors can anatomically integrate into the outer retina and express photoreceptor-specific markers such as recoverin, rhodopsin, and cone opsin,[19] but there isn't compelling proof that complete photo-receptor differentiation or full photoreceptor function ensues.[20] Although additional research is needed to fully elucidate the potential of stem cell-derived photoreceptor transplantation, this may be the major limiting factor in stem cell-mediated AMD treatment.

Overcoming stem cell concerns

Stem cells offer tremendous potential for future treatment of several ocular and non-ocular degenerative diseases, but there are still many hurdles:

  • The concern that receives the most attention is the ethical dilemma posed by creating hESCs. Much of this debate is fueled by politics and is often decided by who is sitting in the White House. As noted, federal funding is frozen for the creation of new hESC lines but is ongoing for existing lines. The use of iPSCs allows scientists to bypass this ethical concern because it does not involve destruction of human embryos.
  • iPSCs also circumvent the possibility of immune rejection, as they are derived from autologous sources. In contrast, hESCs are derived from exogenous sources, so immune rejection is always a possibility if donors and recipients are not HLA-matched. Local and/or systemic immunosuppression must be considered when transplanting hESC-derived cells.
  • Tumorigenesis is a potential concern for both ESCs and iPSCs. Tumors, especially teratomas, may form when undifferentiated precursor cells are not separated from culture and are injected with the differentiated cells. Arnhold et al discovered a 50% incidence of subretinal tumor formation in mouse eyes at 2 months after ESC transplantation.[21] In contrast, another study utilizing 18 different hESC lines showed no evidence of tumor formation.[22] With improved techniques in selecting only the differentiated cells in culture, tumorigenesis in recent studies is quite rare.
  • Stable integration of RPE cells and photoreceptors into host tissue is difficult in disease models such as AMD because of damage to other important structures. For example, Bruch's membrane tends to degenerate in advanced forms of AMD; thus, newly transplanted RPE and photoreceptor cells do not have a healthy scaffold on which to build. The RPE cells then have difficulty polarizing and forming a monolayer. It is evident that future stem cell therapy for atrophic age-related macular degeneration will have to address this issue. Scientists have developed synthetic polymers to serve as a Bruch's membrane substitute in animal models and have achieved successful RPE cell transplantation as a result.[23] [24] These polymers must not only provide a scaffold for RPE cell growth and proper alignment, but also must allow transport of oxygen and nutrients from the choriocapillaris to the RPE and outer retina.

What's ahead for stem-cell therapy?

'Stem cell technology is advancing rapidly; it is possible we will be able to take a skin sample from an AMD patient and create a self-renewing line of healthy RPE and photoreceptor cells for subretinal transplantation and restoration of visual function.'

While tremendous growth has been seen over the past 10 years in developing new AMD treatment strategies, the upcoming decade may hold even more promise. Stem cell technology is advancing rapidly; it is possible we will be able to take a skin sample from an AMD patient and create a self-renewing line of healthy RPE and photoreceptor cells for subretinal transplantation and restoration of visual function.

Stem cell therapy could be implemented in atrophic AMD treatment primarily in 2 ways:

  1. Rescue of diseased retinal cells by secretion of growth and neuroprotective factors, and
  2. Replacement of dead cells by transplantation and functional integration into host tissue.

Stem cell therapy could also be used to treat exudative AMD after stabilization of choroidal neovascularization (CNV) with anti-VEGF therapy. Submacular surgery could be reintroduced in the treatment of exudative AMD. Stem cell-derived RPE and photoreceptor transplants would theoretically be able to "fill in" RPE and photoreceptor defects generated by the surgery, assuming the difficulties associated with synaptic integration can be overcome.

Although many challenges still need to be overcome before stem cell therapy can benefit AMD patients, significant advances have recently been achieved. Human clinical trials are under way for stem cell-mediated treatment of Stargardt's macular dystrophy, and the FDA has just approved a clinical trial for stem cells in the treatment of dry AMD. Advanced Cell Technology, Inc of Santa Monica, California, received FDA approval in January 2011 to implant hESC-derived RPE cells in the subretinal space of 12 patients with atrophic AMD. It is likely only a matter of time before stem cells become an important part of our armamentarium in the treatment of AMD. I

References

  1. Marchetti V, Krohne TU, Friedlander DF, Friedlander M. Stemming vision loss with stem cells. J Clin Invest. 2010;120:3012-3021.
  2. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-1147.
  3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;16:493-503.
  4. Kim D, Kim CH, Moon JI et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009;4:472-476.
  5. Li W, Wei W, Zhu S et al. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell. 2009;4:16-19.
  6. Age-Related Eye Disease Study Research Group: A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration. AREDS report no. 8. Arch Ophthalmol. 2001;119: 1417-1436.
  7. Zhang X, Bok D. Transplantation of retinal pigment epithelial cells and immune response in the subretinal space. Invest Ophthalmol Vis Sci. 1998;39:1021-1027.
  8. Algvere PV, Gouras P, Dafgård Kopp E. Long-term outcome of RPE allografts in non-immunosuppressed patients with AMD. Eur J Ophthalmol. 1999;9:217-230.
  9. Joussen AM, Joeres S, Fawzy N, et al. Autologous translocation of the choroid and retinal pigment epithelium in patients with geographic atrophy. Ophthalmology. 2007;114:551-560.
  10. Cahill MT, Freedman SF, Toth CA. Macular translocation with 360 degrees peripheral retinectomy for geographic atrophy. Arch Ophthalmol. 2003;121:132-133.
  11. da Cruz L, Chen FK, Ahmado A, Greenwood J, Coffey P. RPE transplantation and its role in retinal disease. Prog Retin Eye Res. 2007;26:598-635.
  12. Osakada F, Ikeda H, Mandai M, et al. Toward the generation of rod and cone photoreceptors from mouse, monkey, and human embryonic stem cells. Nat. Biotechnol. 2008;26:215-224.
  13. Haruta M, Sasai Y, Kawasaki H, et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest Ophthalmol Vis Sci. 2004;45:1020-1025.
  14. Lund RD, Wang S, Klimanskaya I, et al. Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells. 2006;8:189-199.
  15. Lambda DA, Gust J, Reh TA. Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell. 2009;4:73-79.
  16. Buchholz DE, Hikita ST, Rowland TJ et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 2009;27:2427-2434.
  17. Carr AJ, Vugler AA, Hikita ST et al. Protective effects of human iPSderived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One. 2009;3;4:e8152.
  18. Lu B, Malcuit C, Wang S et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells. 2009;27:2126-2135.
  19. Klassen HJ, Ng TF, Kurimoto Y et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol Vis Sci. 2004;45:4167-4173.
  20. MacLaren RE, Pearson RA, MacNeil A et al. Retinal repair by transplantation of photoreceptor precursors. Nature. 2006;444:203-207.
  21. Arnhold S, Klein H, Semkova I, Addicks K, Schraermeyer U. Neurally-selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space. Invest Ophthalmol Vis Sci. 2004;45:4251-4255.
  22. Gullapalli VK, Sugino IK, Van Patten Y, Shah S, Zarbin MA. Impaired RPE survival on aged submacular human Bruch's membrane. Exp Eye Res. 2005;80:235-248.
  23. Thumann G, Viethen A, Gaebler A et al. The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes. Biomaterials. 2009;30:287-294.
  24. Lee E, Maclaren RE. Sources of RPE for replacement therapy [published online ahead of print July 3, 2010] . Br J Ophthalmol. 2011;95:445-449.

For reprint permission and information, please contact Jill Blim: jill.blim@asrs.org.

Author affiliations

Sachin Mehta, MD
Retinal Consultants of Arizona, Ltd.

Posted May 31, 2011

Financial disclosures

Dr. Mehta has no relevant disclosures.