Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

Zhongzhong Tong

Department of Ophthalmology and Visual Sciences, Moran Eye Center and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

Vanesa Torbidoni

Facultad de Ciencias Biomédicas, Universidad Austral, Pilar B1629AHJ, Buenos Aires, Argentina, 54-2322-482959, 54-2322-482205 atorbidoni@cas.austral.edu.ar

Yumiko Umino

Center for Vision Research, Department of Ophthalmology, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA

Vaegan

School of Optometry, University of New South Wales, Australia

Krisztina Valter

CNS Stability and Degeneration Group and ARC Centre of Excellence in Vision Science, Research School of Biological Sciences, The Australian National University, 02 6125 1095, 02 6125 0758

valter@rsbs.anu.edu.au

Amit Vasanji

Lerner Research Institute, Cleveland Clinic Foundation, Case Western Reserve

University, Cleveland, Ohio

Vidyullatha Vasireddy

Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, 734-647-6330, 734-936-7231

vasired@umich.edu

Camasamudram Vijayasarathy

Section for Translational Research in Retinal and Macular Degeneration, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA

camasamudramv@nidcd.nih.gov

Tiffany A. Walker

Rehab R&D, Atlanta VA Medical Center, Decatur, GA 30033, USA

Changguan Wang

Department of Ophthalmology and Visual Sciences, Moran Eye Center and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

Dana Wanner

Unit of Gene Therapy and Stem Cells Biology, Hôpital Ophtalmique Jules-Gonin,

Lausanne, Switzerland

Rong Wen

Department of Ophthalmology, University of Pennsylvania, School of Medicine; Philadelphia, PA 19104, 215-746-0207, 215-746-0209 rwen@mail.med.upenn.edu

Andreas Wenzel

Laboratory for Retinal Cell Biology, Dept. of Ophthalmology, University of Zürich, Zürich, Switzerland

Patrick Whalen

Departments of Molecular Genetics and the Center for Vision Science, University of Florida, Gainesville, FL 32610, USA

David S. Williams

Departments of Pharmacology and Neurosciences, UCSD School of Medicine, La Jolla, California, 92093-0983, USA

Zhenglin Yang

Department of Ophthalmology and Visual Sciences, Moran Eye Center and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

Barbara Zangerl

Department of Clinical Studies Philadelphia, School of Veterinary Medicine, University of Pennsylvania Philadelphia. PA 19104

Yong Zeng

Section for Translational Research in Retinal and Macular Degeneration, National Institute on Deafness and other Communication Disorders; National Institutes of Health, Bethesda, MD 20892, USA

Kang Zhang

Department of Ophthalmology and Visual Sciences, Moran Eye Center and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

Yadan Zhang

Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, UK

Jialiang Zhao

Department of Ophthalmology, Peking Union Medical College Hospital, Beijing

100730, China

Lian Zhao

Department of Ophthalmology, University of Pennsylvania, School of Medicine,

Philadelphia, PA 19104

Yu Zhao

Department of Ophthalmology and Visual Sciences, Moran Eye Center and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

Lixin Zheng

Dean A. McGee Eye Institute, Ophthalmology, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd., DMEI 409, Oklahoma City, OK 73104

Part I

Keynote Lectures

Curing Blindness with Stem Cells:

Hope, Reality, and Challenges

Ruben Adler

This article is a summary of a presentation at the XII International Symposium on Retinal Degeneration in Bariloche, Argentina, in October of 2006, in which the author presented his personal views of the contributions that the study of retinal cell differentiation during normal embryonic development can make towards overcoming the limitations that have so far prevented curing blindness through stem cell transplantation. The talk was illustrated mainly with examples derived from research on retinal cell differentiation in the author’s laboratory, and the same approach will be kept in this article; for a broader coverage of the field, the reader is referred to several comprehensive reviews, e.g., (Adler, 2000; Boulton and Albon, 2004; Galli-Resta, 2001; Hatakeyama and Kageyama, 2004; Jean et al., 1998; Livesey and Cepko, 2001; Lupo et al., 2000; Malicki, 2004; Marquardt and Gruss, 2002; Rapaport et al., 2004; Vetter and Brown, 2001; Zhang et al., 2002).

1 Introduction

The death of photoreceptor cells causes visual loss, and eventually blindness, in diseases such as age-related macular degeneration and retinitis pigmentosa, for which currently there is no cure. Stem cell transplantation has emerged as a potential strategy to restore vision in patients affected by these diseases; for this treatment to be effective, the transplanted stem cells would have to differentiate into rod and cone photoreceptors, which would then become synaptically connected with the appropriate cells of the host retina. Despite some promising observations in experimental animals, anatomical and functional stem cell-based restoration of the retina has not been accomplished (e.g., (Ahmad et al., 2004; Banin et al., 2006; Boulton and Albon, 2004; Canola et al., 2007; Chacko et al., 2000; Das et al., 2005; Haruta, 2005; Haynes and Del Rio-Tsonis, 2004; Klassen et al., 2004;

R. Adler

The Johns Hopkins University School of Medicine, Baltimore, Maryland, Tel: 410-955-7589, Fax: 410-955-1749

e-mail: radler@jhmi.edu.

Limb et al., 2006; Meyer et al., 2005; Meyer et al., 2006; Qiu et al., 2005; Suzuki et al., 2006; Takahashi et al., 1998; Warfvinge et al., 2006; Young, 2005), among others). The challenge, then, is to find treatments that would “make stem cells do what we want them to do”; this would require substantial progress in three separate, but closely interrelated areas: (1) how to get “perfect” stem cells; (2) how to induce stem cells to differentiate into rod and cone photoreceptors; and (3) how to induce transplanted stem cells to form synapses with cells in the host retina.

2 How to Get “Perfect” Stem Cells

2.1 What are “Stem Cells”?

Stem cells are cells that (i) can self-renew (i.e., replicate and give rise to new stem cells), and (ii) under appropriate conditions will stop dividing and give rise to two or more (or all) of the specialized cell types of the body. As discussed in some detail below, the developmental potential of stem cells is an issue of great practical relevance, since it becomes progressively restricted during embryonic development and will influence what the cells do when transplanted into a patient. The fertilized egg is totipotent, since it can give rise to all the cell types in the body as well as to the fetal components of the placenta. The cells in the inner mass of the blastocyst (from which the so called “embryonic stem cells” are derived) are pluripotent, that is to say, they can give rise to all the cell types of the body, but not to those of the placenta. The stem cells found in developing organs at later embryonic stages and in the adult are multipotent because they can give rise to two or more, but not to all the cell types of the body (these cells are sometimes referred to as “progenitors”, rather than as “stem cells”). Considerable excitement was triggered by experiments suggesting that, despite their restricted developmental potential, adult stem cells retained a considerable degree of plasticity, and could be “re-programmed” to such a degree that cells from the blood lineage, for example, could give rise to neurons. These apparent examples of “transdifferentiation” have been thrown into doubt by further studies that suggested that they could be artifactual consequences of phenomena such as cell fusion (Corti et al., 2004; Cova et al., 2004; Krabbe et al., 2005).

2.2 Stem Cells and “Cloning”

The concept of “cloning” is used in the field of stem cell biology in three different ways, which deserve separate consideration. Clonal cultures are populations of stem cells derived from a single cell. It is frequently assumed that clonal populations are homogeneous because of their origin; this assumption appears to be frequently unwarranted, however, as illustrated by the finding that among 15,000 cells in a “clonal” culture derived from a single retinal stem cell, fewer than 20 cells had retained stem cell properties (Tropepe et al., 2000). Therapeutic cloning refers to a strategy for generating autologous material for transplanta-

A strategy for inducing stem cells to develop as photoreceptors, therefore, could involve genetic manipulation (e.g., transfection with transcription factors), and/or treatment of the cells with growth factors or similar signaling molecules. Ideally, only one or a small number of these treatments would be necessary to achieve the desired goal of inducing stem cells to develop into perfectly mature, fully differentiated, synaptically connected photoreceptors. “Magic bullets” of this type have not been identified, however, and we don’t yet know how to “make stem cells do what we want them to do”. A possible strategy for overcoming these limitations can be found in the words of Viktor Hamburger, a founding father of Developmental Neurobiology, who said that “Our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right,” [cited in (Purves, 1985)]. In this section I will summarize some of the lessons derived from studies of the dynamics and mechanisms of embryonic development, which are particularly relevant in the context of this article.

3.1 The Embryo does Things in a Sequence

To recapitulate the processes through which photoreceptors are normally generated, transplanted embryonic stem cells would have to go through a series of transitions similar to those that occur during normal development in the embryo, which forms ectoderm before forming a central nervous system (CNS), CNS before forming a retina, a retina before forming photoreceptors, etc. The number of such transitions would obviously be lower if the stem cells are isolated from the retina itself, but considerable complexity can be expected even in this case. The retina initially consists of a population of multipotential, dividing neuroepithelial cells; over time, increasing numbers of neuroepithelial cells become postmitotic (i.e., are “born”), and migrate to different laminar positions within the retina. The future photoreceptors don’t migrate at all, and remain in the future outer nuclear layer. It has been well established in many experimental animals that the differentiation of these cells as photoreceptors occurs in at least two distinct, independently regulated stages

(Bradford et al., 2005; Bruhn and Cepko, 1996; Johnson et al., 2001; Morrow et al., 1998; Stenkamp et al., 1997). The future photoreceptors express a small number of photoreceptor-specific molecules shortly after they are generated, but their “terminal differentiation”, including the expression of most photoreceptor-specific genes, the formation of outer segments and the development of synaptic contacts between photoreceptors and post-synaptic cells, does not start until many days (or even several weeks) after the photoreceptors are generated. During the time interval between photoreceptor generation and terminal differentiation, ganglion cells send their axons to the appropriate brain centers, and the patterning of their terminals in the brain is influenced by electrical activity generated in the inner retina itself (Shatz, 1990a,b). What (if any) influences are exerted by the inner retina upon differentiating photoreceptors is not well understood, and deserves to be investigated, because it appears likely that stem cells transplanted into a degenerating

retina would encounter a microenvironment completely different from that of the developing embryo. This issue will be revisited in the section on synaptogenesis.

The mechanisms that control early and late aspects of photoreceptor differentiation are incompletely understood, but two principles appear well established: (1) the initial developmental program of photoreceptor progenitors controls several important aspects of their differentiation in a cell-autonomous manner, and (2) key aspects of the terminal differentiation of photoreceptor cells are independently regulated by separate microenvironmental factors. The cell autonomous phase of photoreceptor differentiation became evident in experiments in which chick embryo retinal cells were isolated on embryonic days 5–8, before the onset of their overt differentiation, and cultured in an artificial, homogeneous microenvironment, [rev. (Adler, 2000; Adler, 2005)]. The cultured cells appeared homogeneous and undifferentiated at culture onset, but over time some of the cells developed as photoreceptors, and others as non-photoreceptor neurons (predominantly, amacrine cells). Isolated photoreceptor progenitors expressed a fairly complex set of photoreceptor-specific properties in a cell-autonomous manner. The progenitor cells initially had a circular outline, but they subsequently elongated markedly and developed structural and molecular polarity, accompanied by formation of a small outer segment-like process, the expression of several photoreceptor-specific genes, and functional activities including photomechanical responses to light.

On the other hand, a very large number of genes must be expressed in concert during the terminal differentiation of photoreceptor cells, and they are regulated through different mechanisms. These molecules include not only visual pigments, but also phototransduction cascade components, structural proteins (such as those required for outer segment and synapse assembly and maintenance), molecules related to vitamin A transport, neurotransmitters, etc. Several different experimental treatments were shown to selectively induce or repress the expression of particular visual pigment genes in cultures of chick retina photoreceptors (Adler et al., 2001; Belecky-Adams et al., 1999; Bradford et al., 2005; Xie and Adler, 2000). This made it possible to investigate whether those changes were coordinated with changes in the expression of other rodor cone-specific genes; the results showed convincingly that they were not (Bradford et al., 2005). Rather, the data showed that visual pigments and other photoreceptor-specific genes were not co-expressed during development, were not necessarily co-induced, and were not necessarily co-repressed. Taken together, these results demonstrate that the differentiation of photoreceptor cells is a multi-step process that appears to be regulated one step at a time, suggesting that it is unlikely that a single “magic bullet” will be found which would control the entire process of differentiation of a stem cell into a mature photoreceptor.

3.2 Molecular Recycling During Embryonic Development

Many intracellular transcription factors and extracellular signaling molecules are used time and again in different regions of the embryo and at different

stages of development, and their roles are frequently different, or even opposite, depending on the molecular context in which they act. The changing roles of the transcription factor Pax6 in photoreceptor development provide an example relevant to the topic of this article, because (i) the embryo does not form a retina without Pax6, and therefore can not generate photoreceptors, but (ii) photoreceptors will not develop if Pax6 is not down-regulated after the retina is formed.

Pax6 is expressed in both the optic vesicle and the lens ectoderm during early embryonic development, and its mutations cause complex ocular malformations such as aniridia and small eye (Glaser et al., 1994; Glaser et al., 1992; Hanson et al., 1994; Hill et al., 1991a,b; Matsuo, 1993). It has not been conclusively established whether Pax6 is normally necessary in both the optic vesicle and the lens epithelium; until recently, however, the prevailing view was that it is only needed in the ectoderm (Ashery-Padan and Gruss, 2001; Drueppel et al., 2004; Mathers and Jamrich, 2000; Ogino and Yasuda, 2000). This was re-examined in our laboratory by electroporating antisense oligonucleotide morpholinos into the chick embryo optic vesicle (Canto-Soler and Adler, 2006). Electroporation of a control morpholino did not interfere with eye development. On the other hand, a morpholino against Pax6 caused Pax6 downregulation, as expected, and this was followed by a marked increase in cell death in the optic vesicle neuroepithelium. As the embryo continued to develop, the neural retina failed to develop and, despite the fact that Pax6 expression in the lens was normal, lens development was also abortive. The experiment demonstrated that Pax6 expression in the optic vesicle was needed for retina development, and that Pax6 expression in the lens epithelium was not sufficient to support normal lens development. The experiments demonstrated, moreover, that Pax6 is critically needed in the optic vesicle during a very narrow time window: the phenotype was very severe when Pax6 morpholinos were electroporated at or before Hamburger Hamilton stage 10 (Hamburger and Hamilton, 1951), but it was quite mild when electroporation was performed at or after stage 11. It is noteworthy that only 7 hours separate stages 10 and 11. This example illustrates a general feature of experimental embryology, which is that the consequences of a treatment depend not only on its nature, but also on the time (developmental stage) at which it is applied.

Pax6 is initially expressed quite broadly across the undifferentiated retinal neuroepithelium; upon further development, however, Pax6 is downregulated in photoreceptor progenitors, while remaining strongly expressed in prospective amacrine and ganglion cells (Belecky-Adams et al., 1997). This led us to hypothesize that Pax6 could be both a positive regulator of amacrine and ganglion cell differentiation, and a repressor of photoreceptor development. This hypothesis was tested by experimental over-expression of Pax6 into retinal progenitor cells in culture. As predicted by this hypothesis, Pax6 decreased significantly the frequency of progenitor cells that develop as photoreceptors, while promoting their differentiation as amacrine and ganglion neurons (Toy et al., 2002). Ongoing experiments have shown similar effects of Pax6 over-expression in vivo (Canto Soler and Adler, unpublished). In addition to Pax6, other transcription factors, including Chx10 and