SPECIAL ARTICLE Therapy for Ocular Disorders

Leonard A. Levin, MD, PhD; Robert Ritch, MD; Julia E. Richards, PhD; Teresa Borra´s, PhD

ell injury or degeneration occurs in a number of blinding diseases. Therapy has clas- sically consisted of preventing the initial injury or increasing the resistance of cells to injury (cytoprotection). Recently, it has become possible to repopulate tissue com- partments with stem cells. This article presents a current summary of ocular stem cellC research and applications to disease. It is based on presentations and discussions from the July 2002 international conference “Stem Cells and Glaucoma” sponsored by the Glaucoma Founda- tion. This meeting, the first of its kind, brought together ophthalmologists, geneticists, immunolo- gists, and developmental biologists working on stem cell development and applications in both human and animal models. Arch Ophthalmol. 2004;122:621-627

Stem cells are undifferentiated cells able sitates that we first gain an understanding to divide indefinitely yet maintain the abil- of their proliferation, migration, differen- ity to differentiate into specific cell types. tiation, immunogenicity, and establish- They are able to survive throughout the ment of functional cell contacts.2 It will also lifetime of the organism, while maintain- be necessary to produce these cells in con- ing their number, producing populations ditions that meet appropriate safety and of daughter cells (transit amplifying cells) effectiveness standards. Our current un- that can proceed down unique pathways derstanding of the critical factors affect- of differentiation. Stem cells may be ob- ing stem cell behavior remains limited. tained from embryonic tissues, umbilical Rapid progress is being made, and some cord blood, and some differentiated adult of the first applications of stem cells to tissues. Although the potential for stem wound repair in human eyes have pro- cell–based therapies for a variety of hu- duced successes that offer hope for the use man diseases is promising, numerous of stem cells in other ophthalmologic con- problems remain to be overcome, such as ditions. In this article, we discuss current methods for obtaining, transplanting, in- concepts in stem cells and the eye and ducing differentiation, developing func- evaluate stem cell therapy in glaucoma as tion, and eliminating immune reactions.1 a paradigm for novel approaches to the Stem cells have great potential value in treatment of eye disease. treating eye diseases characterized by ir- reversible loss of cells, such as glaucoma SOURCES OF STEM CELLS and photoreceptor degeneration. Although stem cells offer great op- The best understood stem cells are em- portunities for repair of the nervous sys- bryonic stem cells, which derive from early tem and the eye, their clinical use neces- fetal development. To our knowledge, hu- man embryonic stem cells were first char- From the Department of Ophthalmology and Visual Sciences, University of Wisconsin, acterized in 1998.3 These cells are plu- Madison (Dr Levin); Department of Ophthalmology, New York Eye and Ear Infirmary, ripotent (able to differentiate into a wide New York (Dr Ritch); Department of Ophthalmology, New York Medical College, Valhalla (Dr Ritch); Department of Ophthalmology and Visual Sciences, W. K. Kellogg variety of cell types) and relatively easy to Eye Center, University of Michigan, Ann Arbor (Dr Richards); and Department of maintain in culture, but they are neces- Ophthalmology, University of North Carolina, Chapel Hill (Dr Borra´s). The authors sarily allogeneic (from a different genetic have no relevant financial interest in this article. The Glaucoma Foundation meeting donor) to the potential recipient. Embry- attendees are listed in a box on page 626. onic stem cells are continuous cell lines

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 and have the potential to differen- of unilateral disease or allografts Embryonic stem cells are available, tiate into retinal neurons, such as from relatives or cadaver eyes for bi- but there is a substantial risk that if photoreceptors, so they might serve lateral disease. Recently, cultured transplanted into the chamber angle, as an inexhaustible source of neu- limbal stem cells have been used; a they will not differentiate but rather ral progenitors for stem cell therapy small biopsy specimen from a continue to proliferate in the cham- in the retina. Adult stem cells, as healthy limbus can be expanded ex ber angle. This would worsen, rather the name implies, are derived from vivo and then grafted to an eye with than improve, outflow. It is there- mature organisms and are present stem cell deficiency.7 Systemic im- fore necessary to identify mecha- only in restricted cellular compart- munosuppression is necessary in all nisms to induce differentiation of ments.4 They are multipotent (able cases in which allograft limbal stem embryonic stem cells to cells that to differentiate into a restricted num- cells are transplanted, although some express the trabecular meshwork ber of cell types). Stem cells de- patients may eventually achieve a phenotype. rived from the central nervous sys- state of immunologic tolerance and tem (CNS) and ocular tissues have immunosuppression can be discon- DIFFERENTIATION been identified as sources for cells tinued. Future studies will focus on OF STEM CELLS that may someday be used to repair the potential use of adult pluripo- damaged brain, spinal cord, and tent stem cells for ocular surface re- To obtain large numbers of en- retina. Stem cells within the eye have construction and also strategies for grafted stem cells that differentiate received attention because of the promoting a state of tolerance in al- in a desired way, strategies are possibility that they could be ob- lograft limbal transplantation.8,9 needed to channel cells into de- tained from a patient with eye dis- Autologous conjunctival bi- sired phenotypes.15 Modification of ease and used autologously. opsy specimens obtained in the su- the microenvironment and/or inhi- perior fornix, where conjunctival bition of intracellular signaling Retinal Stem Cells stem cells reside, can be expanded cascades in engrafted cells will in tissue culture by using amniotic be needed for appropriate cell- Stem cells have been discovered at membrane as a carrier and can then specific differentiation into injured the pigmented ciliary margin of the be surgically transplanted to the ocu- tissue.16 adult mouse retina.5 A mouse eye lar surface. These tissue equiva- There are several likely sources contains about 100 of these cells, lents have been used successfully in of neural progenitors with retinal po- while the human eye contains about conjunctival replacement after pte- tential that may make stem cell 10000. They can be isolated from rygium surgery and for repair of therapy for eye disease possible.17 eye bank eyes, even from elderly pa- leaking from a scarred filtering bleb. Progenitors isolated from later stages tients. Retinal stem cells do not dif- This use of conjunctival stem cells of retinal development, which nor- ferentiate to form brain cells yet are suggests additional future applica- mally do not give rise to retinal gan- capable of producing all of the dif- tions for these tissue equivalents. glion cells (RGCs), can develop into ferent retinal cell types. Although The corneal endothelium may RGCs in conducive conditions.18 human brain stem cells grow slowly, contain regions for storage (most pe- These cells extend processes to tec- retinal stem cells require no growth ripheral), regeneration (paracen- tal explants (a target for the RGC factors and grow easily and rap- tral), and migration of stem cells. An axon) and appear to function like idly, even in completely defined se- area of corneal endothelial cells ad- RGCs. Stem cells derived from adult rum-free media. Retinal stem cells jacent to the Schwalbe line may be pigmented ciliary epithelium are an can also be isolated from fetal reti- able to transit amplifying cells and excellent source of retinal progeni- nas.6 Both types of retinal stem cells slow-cycling cells.10 Endothelial cell tors because they can differentiate could lead the way for stem cell ocu- density is markedly increased in this along photoreceptors and RGC lin- lar therapies, such as implanting area, as compared with central en- eages.19 Another readily accessible photoreceptors grown in culture into dothelial cell density.11,12 and promising source of neural pro- the blind eye of an individual with Although cell division in the genitors for autologous stem cell retinitis pigmentosa or other reti- normal primate trabecular mesh- therapy is the adult limbal epithe- nal degenerative disorders. work is rare, a niche for trabecular lium (also discussed in the earlier meshwork stem cells might exist at subsection on anterior segment stem Anterior Segment Stem Cells the Schwalbe line in monkeys. Cells cells). Although nonneural in ori- at the Schwalbe line appear differ- gin, progenitors from limbal epithe- Limbal stem cells located in the basal ent from trabecular meshwork cells, lium can generate both neurons and limbal area are involved in renewal and cells with a similar phenotype glia.20 of the . Defi- to the former seem to migrate to the The influence of the age of the ciency due to aniridia, chemical trabecular meshwork.13,14 Transplan- host on the fate of stem cells after burns, Stevens-Johnson syndrome, tation of trabecular meshwork stem transplantation has been studied in or pemphigoid leads to conjuncti- cells to glaucomatous eyes might im- the Brazilian opossum, a small valization, neovascularization, scar- prove aqueous outflow. Theoreti- pouchless marsupial whose young ring, and ulceration of the . cally, trabecular meshwork stem are born in an immature state.21 Limbal stem cells can be trans- cells might be isolated from adult or Brain progenitor cells from mice ex- planted by using autografts in cases embryonic trabecular meshwork. pressing green fluorescent protein as

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 a marker were transplanted via ture (postmitotic) retina, this trans- induced neurogenesis in the fish intraocular injection into develop- formation is difficult to achieve. retina is due to the presence of stem ing and mature opossum eyes.22 Local microenvironmental cues in- cells that perpetually reside in this These cells differentiated in host fluence phenotypic differentiation of tissue. Studies are under way to iden- retinas, often displaying morpholo- grafted cells. Retinal stem cells de- tify the genes expressed by retinal gies characteristic of RGCs, ama- rived from mice expressing green stem cells and the molecules that crine cells, bipolar cells, and hori- fluorescent protein can develop into regulate their neurogenic activity, zontal cells. Transplanted cells photoreceptors and bipolar cells in both during normal growth and af- generally followed the architectural vitro and in vivo.28 With the devel- ter injury.31,32 Knowledge of how organization of the host eyes. The opment of tissue engineering, reti- these cells maintain their ability for greatest morphological integration nal stem cells impregnated into poly- neurogenesis may eventually be ap- and differentiation was observed mers might be grafted into the plicable to human cells. in the youngest host eyes, with subretinal space. little integration in mature eyes. spheres have STEM CELLS AND GLAUCOMA Transplanted brain progenitor cells been injected into the vitreous of may be capable of responding to DBA/2J mice, which have heredi- The only treatment proved for glau- local microenvironmental cues that tary pigmentary glaucoma (M. J. coma is pharmacological or surgi- promote their differentiation and Young, PhD, et al, unpublished data, cal lowering of the intraocular pres- integration. 2003). In 14-month-old mice with sure, but disease in many patients Gene expression analysis can be mild depletion of their RGCs, some progresses despite treatment. Even used to determine the genes in- transplanted cells entered the retina, if new treatments were developed volved in the transition from the mul- elaborated processes, expressed neu- that could stop all future develop- tipotent to the differentiated state. rofilaments, and actually sent pro- ment of visual field loss, there would New retinal cells are continually cesses into the plexiform layer. At 4 still be a substantial need to deal with added at the ciliary marginal zone in months after transplantation, fi- the profound visual field losses in fish and amphibians,23 and it is pos- bers were seen entering the optic millions of people who have had sible to study the underlying molecu- nerve head. glaucoma in the past. Neuroprotec- lar mechanisms of stem cells by com- tion of RGCs and their axons33 is an paring gene expression profiles STEM CELLS IN LOWER alternate treatment being investi- between undifferentiated and differ- ANIMALS gated in randomized controlled entiated states or between multipo- trials.34 Other potential treatments tent and nonmultipotent states.24-26 Studies of stem cells in lower ani- for glaucoma are vaccination with mals may provide insights to their antigens that can induce protective STEM CELLS IN INJURED EYES application in mammals. The Dro- autoimmunity35 and improvement of sophila melanogaster ovary pro- ocular blood flow.36 Tissue injury provides a host of fac- vides an attractive model to study There are at least 3 potential tar- tors that influence the fate of im- stem cell biology because both stem gets for stem cell therapy in glau- planted stem cells and restricts their cells and their surrounding cells have coma: the RGC, the optic nerve head, terminal lineages.16 For example, been well defined.29 Many stem cell and the trabecular meshwork. So far, adult rat hippocampal neuronal pro- properties and relationships to their most work has focused on replacing genitor cells have been trans- microenvironments, or niches, can RGCs because their death is the final planted into the vitreous of glauco- be effectively studied at molecular common pathway for visual loss in matous rats in the hope that they and cellular levels. This Drosophila glaucoma and other optic neuropa- would repopulate the retina as system revealed critical issues in thies. Because human RGCs are mam- RGCs. Some of the progenitor cells stem cell research, including the im- malian CNS neurons that cannot di- injected into the vitreous ex- portance of the niche within which vide and differentiate to replace other pressed the neuronal tissue- the stem cells and daughter cells dif- cells lost from disease, blindness from specific microtubule-associated pro- ferentiate or remain unchanged, the glaucoma is irreversible. Finding a tein 2, which suggests that they start ability to identify individual gene way to differentiate stem cells into to develop into a neuronal pheno- products of importance to differen- RGCs and allow them to connect to type (D. S. Sakaguchi, PhD, et al, un- tiation status, and the opportuni- their appropriate targets would be a published data, 2003). ties to use bioinformatics to apply major step in repopulating the neu- Adult human neural progeni- findings from a model to the study rons lost in glaucoma. The main is- tor cells grafted to diseased hosts can of humans. sues to be resolved are survival and express mature neuronal markers, As a fish grows, so does its CNS, differentiation of the stem cell, main- send processes to the appropriate including the retina. Retinal growth taining the state of the surrounding plexiform layer, and extend neu- is partly due to the continual gen- microenvironment, extension of rites into the optic nerve.27 In spe- eration of new neurons. Further- axons into the optic nerve, establish- cific developmental and injury con- more, unlike injury in human ner- ment of functional connections in the ditions, brain-derived cells can vous tissue, injury to the fish retina lateral geniculate nucleus, and appro- differentiate into cells similar to reti- is repaired by regenerative neuro- priate activation of transsynaptically nal neurons. However, in the ma- genesis.30 Persistent and injury- connected cortical targets.

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 The RGC precursor cells intro- junctiva, corneal endothelium, and Microenvironment of the duced into the retina extend pro- retina. One question that has not yet Transplanted Stem Cell cesses into the optic nerve head.27 been addressed is whether stem cells The need to establish a functional are needed at all, or whether at least Another critical issue is that of the network communicating informa- some problems in either the optic environment into which replace- tion to the brain makes the prob- nerve head or the trabecular mesh- ment cells will be introduced. The lem of stem cell replacement of work could be solved by simply processes by which stem cells settle, RGCs especially complex. How- transplanting young healthy differ- differentiate, and extend axons in an ever, because patients lose a sub- entiated cells into the damaged eye. adult eye do not recapitulate what stantial portion of their RGCs be- Another approach to improv- happens during development, and fore developing functional deficits, ing the local environment could come the environment into which they are there is hope that a limited amount through genetic modification of cells introduced may be hostile, as com- of restoration might have a large before they are introduced. Success- pared with the environment in effect on visual capability. ful use of such approaches will re- which the original developmental Efforts to repair the trabecular quire more work on determining processes took place. Restricted abil- meshwork could theoretically im- which genetic modifications are ity of neural implants to survive, mi- prove intraocular pressure regula- needed and also on overcoming prob- grate, and reestablish neuronal con- tion. Restoration of the entire trabec- lems with gene silencing that can hap- nections with the host environment ular meshwork might not be needed, pen not only after differentiation but has limited the success of neural and simple replacement of corneo- also in some cases in the course of pas- transplantation. It is unclear whether scleral cells might suffice because jux- saging cells as they are being grown the postnatal trophic support is suf- tacanalicular cells are not depleted in for use. In the end, it seems likely that ficient to maintain stem cell sur- late stages of glaucoma.37 Although some type of “nurse” cells that clean vival41 or whether scaffolding such eye bank eyes might constitute a up the environment or provide sup- as glial cells or extracellular matrix plentiful source of trabecular mesh- porting factors will need to precede will adequately protect the new work cells, it remains to be seen or accompany the primary cells be- axons that are attempting to make whether rejection of allogeneic cells ing restored. In addition, protection connections and transmit im- would be a problem. of the fragile new cells may also arise pulses. Pretreatment of the injured A third target for cellular reple- from work on a vaccination ap- tissue with growth factors such as tion is the optic nerve head, which proach to recruitment of cells from the neurotrophins or repletion of sup- undergoes substantial remodeling immune system with neuroprotec- porting cells such as astrocytes or and biochemical change in glau- tive functions. other cells producing trophic or dif- coma. Issues to be dealt with in the ferentiating factors could greatly as- region of the optic nerve head in- CHALLENGES IN USING STEM sist the survival of the new cells in clude excavation, activation of as- CELLS FOR EYE DISEASE a previously hostile environment. trocytes, secretion of nitric oxide, The ability of transplanted neural vascular complications, and loss of Safety graft cells to migrate and integrate cellularity.38-40 Progression of glau- into the host retina can be influ- comatous optic neuropathy in the There are strict rules for the condi- enced by intrinsic properties of im- presence of what appears to be clini- tions in which stem cells must be plant cells, as well as by factors in cally adequate lowering of intraocu- grown if they are destined for thera- the host retinal environment. lar pressure may reflect structural peutic use. Cells must be grown and functional changes of optic without serum and without the use Continued Progression nerve head cells. Repopulating these of cell feeder layers, something that of Disease cells with stem cell–derived nor- would potentially complicate main- mal astrocytes and fibroblasts might tenance of at least some stem cell Not only will the differentiated stem be an alternate therapy for glau- types that require other cell types in cell face a hostile environment after coma—one that does not require their local niche to maintain an in- transplantation, but it also may be just complex axonal pathfinding. termediate state of differentiation. as susceptible to the disease as the The optimal source of stem cells With the substantial amount of work original cell. For example, a re- for a particular therapy is a major is- required for approval to use a given populated RGC in an eye with ad- sue. Not all stem cell types can be cell line, it will be essential that such vanced glaucoma may not survive induced to differentiate into all of the cells can be grown in large amounts because of the underlying patho- cell types needed to treat glau- while maintaining a particular stage physiology of the disease. If it took coma. It is encouraging that a num- of multipotent development. In ad- many years for the adverse environ- ber of different sources and types of dition, the cells must differentiate in ment to kill the RGCs, the newly stem cells and precursor cells have completely defined conditions, their transplanted RGCs might survive been identified from which rel- proliferation after transplantation longer, and the restoration of optic evant cell types for ocular stem cell must be shut down, and they must nerve function could last many therapy can be derived. These in- perform the desired functions while years. If not, the restored cells clude not only fetal stem cells but remaining localized to their site of might soon die unless steps are first also cells from brain, limbus, con- targeting. taken to correct the environment,

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 which is a more complex problem. from subependymal regions. The ap- There are at least 5 different Thus, the problem of preexisting in- parent failure of ongoing remyelin- areas in which progress needs to be jury in a diseased eye necessitates that ation in multiple sclerosis could re- achieved. First, more needs to be the damage from the surrounding en- flect a number of factors, including known about precursor cells for vironment be repaired in addition to exhaustion of progenitor cells, lack of any given cell type and about their replacing the primary cells of interest. trophic signals, injured axons being production of essential factors, unreceptive to remyelination, and se- which may mean identification of Pathfinding lective immune injury of the progeni- stem cells that can differentiate into tors.47,48 These issues will need to be cell types that can live (eg, in the The optic nerve carries axons from understood for effective remyelin- optic nerve head) and produce the RGCs to targets in the lateral genic- ation of the optic nerve. appropriate factors. Alternatively, it ulate nucleus in a retinotopic map- may mean genetic modification of ping. For the injured optic nerve to Immune Response cells to achieve expression of genes be restored, there has to be pathfind- encoding such factors. Desired ing of transplanted retinal stem cells Allogeneic stem cells are potential tar- functions include interfering with within the retina that migrate to the gets for the immune system, and their apoptosis, inhibiting factors in appropriate cellular location and send use may be hindered by humoral or amacrine cells, and production of axons to the optic nerve head, then cell-mediated rejection. This may be trophic factors. through the nerve, 50% crossing at the tempered by the relatively immune- Second, much more needs to be chiasm, and eventually to a correct lo- privileged nature of the eye. Al- known about potential sources of cation in the lateral geniculate though the existence of stem cells in stem cells that could be developed nucleus.42,43 Retinal glial cells, includ- the adult eye or other organs offers the in appropriate conditions and that ing astrocytes and Mu¨ ller cells, are the possibility of bypassing allogeneic re- could be approved for therapy. This guardians of the retinal cell layers. In jection through use of the patient’s includes the need to explore embry- the host retina, they play an essen- own stem cells, such cells might carry onic stem cells, brain stem cells, ocu- tial role in preventing graft cell mi- whatever defect initially predis- lar stem cells, and even the trans- gration and integration. It may be pos- posed the eye to glaucoma and there- plantation of some types of mature sible to guide the migration of fore fail to correct the problem. differentiated cells. transplanted neural stem cells by se- Third, microenvironments lectively manipulating glial cell prop- FUTURE DIRECTIONS need to be identified in which stem erties in the host retinal environ- cells can proliferate; differentiate; en- ment. Ensuring crossing at the chiasm There is ample precedent for re- graft; migrate; and, once they are in and precise arrival at axonal targets search in stem cell therapy for neu- the right state and place, shut down remains a difficult problem.44,45 rodegenerative disease. A good ex- proliferation. This may include the ample is motor neuron disease. In need to understand more about Remyelination animal models of amyotrophic lat- other cell types in the surrounding eral sclerosis, human embryonic environments and on the topo- Not only is it necessary for RGCs to brain–derived cells produce rapid graphic map of the target region. reach their appropriate targets, but and profuse motor neuron growth Fourth, study of naturally re- physiological axonal conduction ve- when engrafted into the ventral horn generating systems such as teleost or locity and energy efficiency also re- of the spinal cord. Transplanted em- chick eye offer the chance to iden- quire the presence of myelin. Our bryonic brain–derived cells acquire tify antagonists for inhibitors of re- knowledge of remyelination in adult immunohistochemical markers of generation in the retina and to iden- human CNS derives from the ex- mature neurons and astrocytes and tify the genes involved in growth and tent, or lack thereof, of remyelin- send axonal processes to the periph- differentiation early in develop- ation observed in multiple sclerosis. ery. This process allows paralyzed ment, when the ability of the mam- This observation raises a number of animals to move their hind limbs and malian retina to regenerate has not issues regarding the potential role of walk. yet been shut down. progenitor cells in replacing injured The same will likely be true for Fifth, even some of the sim- myelin and oligodendrocytes in adult using stem cells for ophthalmic dis- plest experiments on transplanta- human CNS. Remyelination occurs in ease. Precursor cells can settle in the tion of trabecular meshwork cells acute rather than chronic multiple retina, differentiate into RGCs, con- have yet to be performed, and little sclerosis lesions. Oligodendrocyte nect with afferent neurons in the in- is known about any potential stem progenitor cells can be identified in ner plexiform layer, and grow into cells or precursor cells from which regions of active multiple sclerosis le- the optic nerve head. Many details the trabecular meshwork might be sions but without apparent increase need to be worked out about the use revived. in numbers, as compared with find- of such cells to restore the complex ings in normal brain tissue.46 At is- delicate neural network of the eye. SUMMARY sue is whether the remyelinating cells Not only cellular regrowth but also are derived from a pool of glial- establishment of functional connec- Although preliminary results have restricted progenitor cells or from tions to the lateral geniculate body been achieved on many different multipotent stem cells that migrate will be required. fronts in applying stem cell technol-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 2. Cao Q, Benton RL, Whittemore SR. Stem cell re- Glaucoma Foundation Meeting Attendees pair of central nervous system injury. J Neurosci Res. 2002;68:501-510. 3. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. The Glaucoma Foundation held a meeting entitled “Stem Cells and Glau- lines derived from human coma” in Chicago, Ill, July 26-27, 2002. The meeting organizers and modera- blastocysts. Science. 1998;282:1145-1147. tors were Terete Borra´s, PhD (University of North Carolina at Chapel Hill, Chapel 4. Presnell SC, Petersen B, Heidaran M. Stem cells Hill, NC), Leonard A. Levin, MD, PhD (University of Wisconsin, Madison, Wis), in adult tissues. Semin Cell Dev Biol. 2002;13: Julia E. Richards, PhD (University of Michigan, Ann Arbor, Mich), and Robert 369-376. Ritch, MD (New York Eye and Ear Infirmary, New York, NY). Participants in- 5. Tropepe V, Coles BL, Chiasson BJ, et al. Retinal cluded J. Wayne Streilein, MD (President, Schepens Eye Research Institute, Bos- stem cells in the adult mammalian eye. Science. ton, Mass), Theodore Krupin, MD (Clinical Professor of Ophthalmology, North- 2000;287:2032-2036. western University School of Medicine, Evanston, Ill), Derek van der Kooy, PhD 6. Yang P, Seiler MJ, Aramant RB, Whittemore (Professor of Anatomy and Cell Biology, Faculty of Medicine, University of SR. In vitro isolation and expansion of human Toronto, Ontario, Canada), Scott Whittemore, PhD (Professor of Neurologi- retinal progenitor cells. Exp Neurol. 2002;177: 326-331. cal Surgery, University of Louisville School of Medicine, Louisville, Ky), Ryo 7. Ramaesh K, Dhillon B. Ex vivo expansion of cor- Kubota, MD, PhD (Assistant Professor, Department of Ophthalmology, Uni- neal limbal epithelial/stem cells for corneal sur- versity of Washington, Seattle), Young Kwon, MD, PhD (Associate Professor face reconstruction. Eur J Ophthalmol. 2003;13: of Clinical Ophthalmology, Department of Ophthalmology, University of Iowa, 515-524. Iowa City), Michael J. Young, PhD (Director, Minda de Gunzburg Research Cen- 8. Holland EJ, Djalilian AR, Schwartz GS. Manage- ter for Retinal Transplantation, Harvard Medical School, Boston, Mass), Si- ment of aniridic keratopathy with keratolimbal al- mon John, PhD (Associate Investigator, Jackson Laboratories, Howard Hughes lograft: a limbal stem cell transplantation tech- Medical Institute, Bar Harbor, Me), Ting Xie, PhD (Assistant Scientist, Stowers nique. Ophthalmology. 2003;110:125-130. Institute for Medical Research, Kansas City, Mo), Peter Hitchcock, PhD (As- 9. Dua HS, Azuara-Blanco A. Limbal stem cells of the sociate Professor of Ophthalmology and Visual Sciences and Developmental corneal epithelium. Surv Ophthalmol. 2000;44: 415-425. Biology, University of Michigan, Ann Arbor, Mich), Iqbal Ahmad, PhD (Asso- 10. Bedrarz J, Engelmann K. Indication of precursor ciate Professor of Ophthalmology and Pharmacology, University of Nebraska cells in adult human corneal endothelium [ab- Medical Center, Omaha), Donald Sakaguchi, PhD (Associate Professor of Zo- stract]. Invest Ophthalmol Vis Sci. 2001;42(suppl): ology and Genetics, Iowa State University, Ames, Iowa), Jeffrey Rothstein, MD, 5274. PhD (Professor of Neurology and Neuroscience, Johns Hopkins University, Bal- 11. Schimmelpfennig BH. Direct and indirect deter- timore, Md), Dong Feng Chen, PhD (Assistant Professor, Department of Oph- mination of nonuniform cell density distribution thalmology, Harvard University, Boston, Mass), Henry Edelhauser, PhD (Di- in human corneal endothelium. Invest Ophthal- rector of Ophthalmic Research, Emory Eye Center, Atlanta, Ga), Ernst Tamm mol Vis Sci. 1984;25:223-229. (Professor of Molecular Anatomy and Embryology, Department of Anatomy, 12. Amann J, Holley GP, Lee SB, Edelhauser HF. In- University of Erlangen-Nu¨ rnberg, Erlangen, Germany), Jack Antel, MD (Profes- creased endothelial cell density in the paracen- sor of Neurology, Montreal Neurologic Institute, Quebec, Canada), Ali Djalil- tral and peripheral regions of the human cornea. Am J Ophthalmol. 2003;135:584-590. ian, MD (National Eye Institute, Bethesda, Md), Michal Schwartz, PhD (Profes- 13. Lutjen-Drecoll E, Kaufman PL. Echothiophate- sor of Neuroimmunology, Weizmann Institute of Science, Rehovot, Israel), Roger induced structural alterations in the anterior cham- Beuerman, PhD (Scientific Director, Singapore Eye Research Institute, ber angle of the cynomolgus monkey. Invest Oph- Singapore), William W. Hauswirth, PhD (University of Florida, Gainesville), Paul thalmol Vis Sci. 1979;18:918-929. Kaufman, MD (University of Wisconsin, Madison), Erin B. Lavik, ScD (Massa- 14. Lutjen-Drecoll E, Kaufman PL. Long-term timo- chusetts Institute of Technology, Cambridge, Mass), James C. Tsai, MD (Colum- lol and epinephrine in monkeys: morphological al- bia University, New York, NY), Abbot F. Clark, PhD (Alcon Research Ltd, Fort terations in trabecular meshwork and ciliary Worth, Tex), John Donello, PhD (Allergan Inc, Irvine, Calif), and Vincent muscle. Trans Ophthalmol Soc U K. 1986;105(pt Michael Patella, OD (Carl Zeiss Ophthalmic Systems Inc, Dublin, Calif). 2):196-207. 15. Yang P, Seiler MJ, Aramant RB, Whittemore SR. Differential lineage restriction of rat retinal pro- genitor cells in vitro and in vivo. J Neurosci Res. 2002;69:466-476. ogy to eye disease, a tremendous optic neuropathies, before accurate 16. Cao QL, Howard RM, Dennison JB, Whittemore amount of work remains. Some of reassembly of the complex visual SR. Differentiation of engrafted neuronal- restricted precursor cells is inhibited in the trau- the main issues are identification of pathways will be achieved. matically injured spinal cord. Exp Neurol. 2002; the optimal types, es- 177:349-359. tablishment of growth and differen- Submitted for publication February 4, 17. Ahmad I. Stem cells: new opportunities to treat tiation conditions that meet safety 2003; final revision received July 15, eye diseases. Invest Ophthalmol Vis Sci. 2001; and effectiveness standards, and the 42:2743-2748. 2003; accepted August 25, 2003. 18. James J, Das AV, Bhattacharya S, Chacko DM, manipulation of the surrounding en- Corresponding author: Leonard Zhao X, Ahmad I. In vitro generation of early- vironment to allow transplanted cells A. Levin, MD, PhD, Department of born neurons from late retinal progenitors. JNeu- to survive and function. Although Ophthalmology and Visual Sciences, rosci. 2003;23:8193-8203. transplanted neuronal precursors 19. Ahmad I, Tang L, Pham H. Identification of neu- University of Wisconsin Medical ral progenitors in the adult mammalian eye. 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