Seminars in Ophthalmology, 21:1–7, 2006Copyright ©c Taylor & Francis Group, LLCISSN: 0882-0538DOI: 10.1080/08820530500501322

Corneal Stem Cells: Bridgingthe Knowledge Gap

P. CharukamnoetkanokUPMC Eye Center,Ophthalmology and VisualScience Research Center, Eyeand Ear Institute, Departmentof Ophthalmology, Universityof Pittsburgh School ofMedicine, Pittsburgh, PA, USA

ABSTRACT Stem cell research offers hope to countless patients whose con-ditions have heretofore been deemed incurable. Better understanding of stemcell behaviors and functions will lead to insights into biological mysteries en-compassing the fields of angiogenesis, development, tissue homeostasis, woundhealing, and carcinogenesis. Clarity of vision requires smooth ocular surface onwhich the corneal epithelial cells undergo continuous turnover every 3 to 10days. Tragically, many patients are blinded and devastated by severe ocular sur-face diseases due to limbal stem cell deficiency even though, besides opaquecornea, their eyes are otherwise healthy. Corneal stem cell transplantation of-fers hope by creating clear windows for these eyes; unfortunately, the long-termsuccessful outcome remains limited. The nature of corneal epithelial stem cell ispoorly understood, but many circumstantial evidences suggest the presence of“source cells” in the limbal region of the eye. Nonetheless, the precise biomarkerof corneal stem cell remains elusive. The stem cell puzzle can be solved withapplication of the fundamental scientific method—asking salient questions atthe right time and finding answers using keen observations and proper tools.Readily accessibility and structural simplicity of the cornea lend themselvesto study of the stem cell biology. The ability to identify and isolate cornealstem cell will be a gateway to meaningful investigation into its biology. Thisadvance will also have direct impact on improving the efficacy of promisingstem-cell-based therapies, including limbal stem cell transplantation.

KEYWORDS angiogenesis, limbus, review, stem cell, transplantation

INTRODUCTIONStem cells are essential for cellular regeneration and repair leading to main-

tenance of homeostasis in many tissues, including the cornea.1−6 After normalwear and tear or tissue injury, stem cells proliferate to repopulate the damagedtissue.7 The defining characteristics of stem cells are asymmetric self-renewal(ability to simultaneously replicate themselves and produce progenitor cells withhigh fidelity), potency (capacity to differentiate into different cell lineages), andniche (protective microenvironment).8,9

Clarity of vision requires smooth ocular surface on which the corneal epithe-lial cells undergo continuous turnover every 3 to 10 days.10,11 This dynamichomeostasis is orchestrated by the centripetal and circumferential migration of

The author would like to express deepand sincere gratitude to Drs. NirmalaSundar Raj and Eric Lagasse for theircritical review of the manuscript.

Address correspondence to PuwatCharukamnoetkanok, M.D., 203Lothrop Street, Suite 1015, Pittsburgh,PA 15213, USA. E-mail: charpx2@upmc.edu

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the epithelia from the limbus (transition zone betweenthe opaque sclera and the clear cornea).12 The renewalsource of corneal epithelia is believed to congregate inthe basal layer of the limbus.13

Corneal stem cell research has a long and illus-trious history, and now there is a merging of criti-cal insights with sophisticated new research tools thatmake future study of the stem cell biology an excit-ing endeavor. Detail deliberation of issues pertinentto limbal stem cells has been accomplished by manyexcellent reviews.2,14−20 This paper will present nu-merous compelling circumstantial evidences suggest-ing the existence of corneal epithelial stem cells, de-scribe current treatment options for limbal stem celldeficient diseases, and discuss future directions forstem cell research that offers unprecedented oppor-tunities to discover novel therapies for debilitatingdiseases and new ways to explore fundamental ques-tions of biology. I will posit that there is an urgentneed to bridge several gaps between the current knowl-edge and the missing information, between the elusiveand the quantifiable properties of stem cells, and fi-nally, between the clinical success and their mechanisticexplanations.

EVIDENCES FOR LIMBAL STEM CELLSPrecise identification, isolation, and characterization

of the stem cells have not yet been possible due tothe current lack of specific markers.19 Active researchefforts are ongoing to search for the elusive limbal stemcells. However, there are many lines of circumstantialevidences suggesting existence of corneal epithelial stemcells in the limbal region. In the succeeding sections,I will discuss some of the most compelling of theseevidences.

CORNEAL EPITHELIUM NEEDSCONSTANT RENEWAL

In 1971, Davanger and Evensen13 observed and re-ported a unique feature of corneal epithelium that theperipheral corneal epithelial cells migrated centripetallytoward the center of the cornea. They speculated thatthere were “source cells” for corneal epitheliums locatedwithin the limbus. While this observation has becomedogmatic, it was a revolutionary proposal at the time ofthe publication. The authoritative textbook at the timestated that the limbus was merely a functionless vestigial

structure. Subsequent works validated the idea that thelimbal cells migrate centripetally and circumferentiallytoward central cornea.2,12,21

XYZ hypothesis22 was proposed to explain the ob-served turnover dynamic of the corneal epithelial cellsduring normal wear and tear (homeostasis) and in re-sponding to injuries. It stated that the amount of thecentripetal migration plus the posteroanterior move-ment of the corneal epithelia must equal to the mag-nitude of cell loss at the ocular surface. As part of thishypothesis, corneal epithelial stem cells are believed tolocalize in the limbal region.

UNDIFFERENTIATED MARKERLOCALIZED IN LIMBAL BASAL CELLS

Corneal epithelial cells synthesized two major tissue-restricted keratins—K3 and K12.23−26 Schermer andcolleagues26 used antibody AE5 to study the expressionof K3 in cultured rabbit corneal epithelial cells as wellas in vivo. They discovered that K3 in vitro expressionlocated mostly in the upper, more differentiated, celllayers suggesting that K3 was a marker for an advancestage of corneal epithelium differentiation. When theresearchers examined the in vivo expression of K3, theydetected expression of this keratin in the upper cell lay-ers of corneal epithelium in the limbal zone. However,they also unexpectedly found expression of K3 in alllayers of the central rabbit corneal epithelium. Basedon this finding, the investigators proposed that cornealstem cells, less differentiated by definition, resided inthe limbal zone instead of uniformly across the entirecorneal epithelial basal layer as had been previouslythought.

Attempting to explain gradual differentiation gra-dient among corneal epithelium, Lavker and Sun4,27

proposed the differentiation scheme of “stem cells” →Transit Amplifying cells (TAC) → terminally differen-tiated cells.

LIMBAL BASAL CELLS HAVE LONGCELL CYCLE TIME AND EXHIBITGREATER DIVISION POTENTIAL

Cells with relatively slow cell cycle can be detectedexperimentally as “label-retaining cells” (LRC).28−31

One of the techniques to identify LRC is the pulse-chase experiment in which one first labels dividing cells

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by perfusing a tissue continuously with tritiated thymi-dine (3HT) or bromodeoxyuridine (BrdU). During achase period, typically 4–8 weeks, the homeostatic fluxof tissue turnover causes the rapidly dividing cells todilute out and lose most of their labels, but the slow-cycling stem cells still retain their labels. Thus, at theend of the experiment, some of the stem cells can bedetected as LRCs.

Several investigators applied this labeling techniqueto mouse corneal epithelium and founded LRCs exclu-sively in the basal layer of peripheral corneal epitheliumin the limbal area.30,32 The central corneal epitheliumlacked such cells.

When different subsets of corneal epithelial cellswere studied in the cell and explant culture, thelimbal cells had a higher in vitro proliferative po-tential than central corneal epithelial cells.33,34 Asimilar observation was also noted in vivo. Lavkerand associates35 found that when mouse limbal andcorneal epithelia were continuously stimulated withphrobol myristate, limbal epithelia maintained a signif-icantly greater proliferative response than the cornealepithelia.

THE PALISADES OF VOGT IS AN IDEALSTEM CELL NICHE IN THE LIMBUSStem cells potentially could last as long as the life

span of the organism. In order to establish and maintaintheir marvelous properties, stem cells must be protectedfrom hostile milieu in a specialized microenvironment,the so-called stem cell niche.8,9 Within this protectiveniche, the stem cells communicate and interact withtheir neighboring cells that organize and secrete extra-cellular matrix and other factors allowing the residentstem cells to manifest and maintain their unique intrin-sic properties.

Nature provides myriad variations in niche de-sign and architecture.36 The hematopoietic stem cells(HSC)37 reside in the bone marrow. Their nichecomposes of many different cell types includingmacrophages, adipocytes, and fibroblasts. Self-renewalHSCs associate with osteoblasts line the inner surfaceof the trabecular bone. The neural stem cells locatein the subventricular zone between the lateral ventri-cle and the stratum. Skin stem cells take up residentin the hair follicle at a region known as the bulge.38

The bulge, receiving inputs from the blood vesselsand sensory nerve endings, is enveloped by a base-

ment lamina, which is surrounded in turn by a dermalsheath.39

The Palisades of Vogt40−43 is a complex structurewith papilla-like columns located in the limbus. Itis richly vascularized and highly innervated. Becauseof these characteristics, the Palisades of Vogt pro-vides uniquely protective microenvironment and ful-fills requirements of the ideal stem cells’ niche in thelimbus.

INJURIES OR SURGICAL REMOVALOF THE LIMBAL REGION RESULTS

IN HEALING WITHNON-CORNEAL EPITHELIA

Normally, the cornea is avascular, but a wide va-riety of insults can cause capillaries invasion formthe limbal vascular plexus. This process of new bloodvessel formation is termed corneal neovascularization(NV).44−46 Three major categories of corneal NV aresuperficial vascularization, fibrovascular pannus, anddeep stromal vascularization. The superficial vascular-ization rarely cause decrease in vision. But the lattertwo types of corneal NV can lead to significant loss ofvision if they involve the visual axis. Corneal NV hasbeen reported in 4.14% of patients (total approximately1.4 million per year) visiting general ophthalmologyclinics in the U.S.47 Worldwide, more than 7 mil-lion people lost their sight due to corneal opacity andvascularizaton.48,49

The precise etiology of corneal NV is an activearea of research.46,50 We will focus on one importantcause of corneal angiogenesis—injured or defective lim-bal stem cells. Corneal epithelial stem cell deficiencyis a debilitating and blinding disease that causes sig-nificant discomfort and morbidity. Etiologies includetrauma (chemical injuries, contact lens wear), inflam-mation (Stevens-Johnson syndrome, ocular cicatricialpemphigoid, severe corneal infection), and congeni-tal (aniridia).51 The hallmark of limbal stem cell de-ficiency is the “conjunctivalization” of the cornea. Inthese diseases, the failure of the epitheliums to prop-erly resurface the cornea triggers a vicious cycle leadingto scaring and vascular invasion. The irony of thesetragic diseases is that, besides opacified and vascular-ized corneas, most patients’ eyes are otherwise healthy.We can rehabilitate these eyes and restore vision by re-plenishing corneal epithelial stem cells and replacing

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damaged corneas with clear “windows” that thrive inhostile diseased environment.

LIMBAL TRANSPLANTS RESULT INREGENERATION OF EPITHELIA WITH

CORNEAL PHENOTYPESince Kenyon and Tseng52 published their ground-

breaking case series, autologous limbal stem cell trans-plantation has proven to be a powerful and effectivesurgical procedure to restore severely damaged ocu-lar surface. However, the harvest of donor tissue in a“blinded” fashion amplifies surgical risks in two ways.If the donor limbal grafts do not contain adequate stemcells, the surgery will either fail immediately or lacklongevity. On the other hand, if too much stem cellsare removed, the donor eye will develop problems as-sociated with limbal stem cell deficiency.53−55

Even more compelling justification for further re-search is the fact that in bilateral diseases the onlysource of stem cells is allogeneic donor tissue—eitherfrom living relatives or cadaveric sources.55−57 Despiteconcomitant administration of powerful immunosup-pressive medications, the long-term success rate of theallogeneic stem cell transplantation remains disappoint-ingly low.58,59

Many novel strategies, such as addition of amnionicmembrane and ex vivo (tissue culture system) expan-sion of the graft,60−62 have been devised to reintro-duce the corneal limbal stem cells. Reports of spectac-ular outcomes during early postoperative period notwithstanding, the success of these procedures does notlast.

The reasons for failure may include inadequate num-bers of stem cells resident within the graft after theharvest, damage or death of stem cells upon place-ment on the inflamed ocular surface, or continuedattrition of stem cells as a result of an inhospitablemicroenvironment. These possibilities have not beenproperly explored due to the inability to precisely iden-tify stem cells. As a result, currently, we must denysight-restoring surgery to many patients who wouldbenefit from limbal stem cells transplantation. In or-der to further improve the safety and efficacy of thetransplantation, we must know mechanistic reasonsfor its success that will require additional advancesin the understanding of the basic biology of stemcells.

FUTURE DIRECTIONS OF CORNEALSTEM CELL RESEARCH

Although stem cell research is on the cutting edge ofbiological science, it is still in its infancy. Accessibilityand structural simplicity of the cornea lend itself to thestudy of stem cell biology. Many candidates for limbalstem cells markers have been proposed (Table 1). How-ever, the true stem cells-specific biomarker is lacking.63

As such, the current knowledge of corneal stem cells islimited. The elusive nature of stem cell marker has beenunderstandably frustrating. Dua provocatively specu-lated that “some might even question the use of theterm stem cell and prefer the term ‘committed progen-itors’ instead.”19

Nonetheless, the first research priority continues tobe identification and characterization of limbal stemcell markers. This accomplishment will allow us to be-gin to build several tools (in vitro assays, and in vivomodels) for investigation of stem cell biology. Further-more, clinically relevant biomarkers will enhance theefficacy of stem-cell-based therapies including limbalstem cell transplantation. The essential question regard-ing the nature of stem cell is what makes stem cell astem cell? Are the regulatory factors intrinsic, extrin-sic, or both? Understanding of stem cell niche is alsoimportant because any therapeutic utilization of stemcells would require harvesting the stem cells, expandingthem in culture, and implanting them into a new tis-sue environment. How do interactions between stemcell and niche component such as extracellular ma-trix and niche resident cells regulate stem cell behav-iors? What other factors—cytokines, angiogenesis fac-tors, neuronal signals, genetic background, and cornealbiomechanics—influence regulation of stem cells? Howcan we overcome the immune-mediated rejection oftransplanted stem cells or their derivatives?

Better understanding of limbal stem cells will en-hance the knowledge of corneal epithelial biology ingeneral. This advance will have significant impact onour comprehension of several unsettled clinical prob-lems, such as recurrent erosion syndrome, persistent ep-ithelial defect, and unpredictable outcomes of refractivesurgery.

Finally, most of the research involving corneal stemcells has traditionally been concentrated on the limbalepithelial stem cells. It would be very exciting to learnmore about the corneal stromal and endothelial stemcells.

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TABLE 1 Cytochemical Differences Between Central Cornea and Limbus

Limbus Central cornea Descriptions References

CK 5/14+ CK 5/14 − Cytoplasmic protein, cytoskeletal intermediatefilament

19

CK 19+ CK 19 −/+ Cytoplasmic protein, cytoskeletal intermediatefilament, marker of undifferentiatedepitheliums

78,79

p63+ p63 − Nuclear Protein, transcription factor 80

ABCG2+ ABCG2 − ATP binding cassette transporters, drugresistance transmembrane protein,? universal SC marker

81−83

Vimentin+ Vimentin − Type III intermediate filament 84

Intrinsic melanogenesis No pigment 19

Cytochrome oxidase and ATPase+ Metabolically inactive Cytoplasmic protein 19

α enolase+ α enolase − Glycolytic enzyme, cell surface protein 85,86

Integrin α9+ Integrin α9 − Cell membrane protein, adhesion molecule 63

Integrin β1 + (strong) Integrin β1 −/+ Cell membrane protein, adhesion molecule 63

EGFR + (stong) EGFR+ Transmembrane protein, receptor 87

CD71 + (bright) CD71+/− Transferrin receptorCK 3/12 − CK 3/12+ Cytoplasmic protein, cytoskeletal intermediate

filament, markers for mature cornealepitheliums.

26

CX43 − CX43+ Membrane protein, gap junction protein 20,88

E-cadherin − E-cadherin+ Intercellular adhesive molecule 89

Integrin α6 − Integrin α6+ Cell membrane protein, adhesion molecule 63

Involucrin − Involucrin+ Early differentiation marker 63

CK: cytokeratin; CX: connexin; EGFR: epidermal growth factor receptor.

STEM CELLS, KERATOPROSTHESIS,AND BIOENGINEERED CORNEA

The field of artificial cornea, keratoprothesis (K-pro),has a long and adventurous history.64−69 A detailednarrative of this important medical history is beyondthe scope of this review. Instead, I wish to brieflysuggest how increased understanding of limbal stemcells might contribute to the progress of bioengineeredcornea.

Dr. Claes H. Dohlman, the father of keratoprosthe-sis, always gracefully insists that since there are so manyunpredictable factors about K-pro, any assumption isvery dangerous.70 The only way to know which de-sign is best for patients is empirical testing in carefullythought-out clinical trials.71−73 There are at least twooverlooked essential corneal functions: the epithelialbarrier function to harmful elements74,75 and being asite of the gold standard way to measure intraocularpressure.76

Since the cornea is a dynamic living tissue, it isnot adequate to simply replace it with a clear pieceof plastic. What are characteristics of an ideal artifi-

cial cornea? It should mimic the optical and biomech-nical properties of a natural cornea but be ableto thrive among inhospitable environments. Bioengi-neered cornea must provide barrier function to tox-ins and infections,77 yet must also be immunologi-cally inert. Postoperatively, there must be an accuratemethod to measure important indices of eye healthincluding the intraocular pressure. Finally, artificialcornea must seamlessly biointregrate into the recipient’seye.

Eons of evolution lead to perfectly resilient and clearcorneas. By incorporating knowledge of stem cells intothe creation of artificial corneas, we will be wisely tak-ing advantage of the force of nature. The challengeis how to apply our knowledge about corneal stemcells to bioengineer an authentic replacement of ca-daveric donor cornea that is truly clinically useful.In order to meet that worthy challenge, much moreresearch effort is needed. Stem cells offer great ther-apeutic potential in a nascent field of regenerativemedicine. Solving the mystery of the stem cell puzzlewill accelerate progress in the bioengineering of thecornea.

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CONCLUSIONThe biology of stem cells is intriguingly complex. We

have only begun to uncover some answers to bridgethe gap in knowledge about this great biological mys-tery. Like Mozart, Bach, or Beethoven, nature can bethought of as a master composer of symphonic multi-cellular organisms. The same theme, stem cell, is variedwithin various tissue types. Elucidation of the behaviorsof the corneal epithelial stem cell will contribute to theunderstanding of other somatic stem cells as well as em-bryonic stem cells. Like the joy of listening to music,one can also readily derives pleasure from witnessingmarvelous biological systems in action. Additionally,just as understanding music theory can enhances one’senjoyment of a symphony, it is truly joyful to learnmore about biological principles underpinning myste-rious actions of stem cells. After all, we may not needto be able to understand all nuances or every complexdetail in order to discover useful insight and share thebenefit of discovery with our patients. That is the ulti-mate reward for all of us.

REFERENCES[1] Blau HM, Brazelton TR, Weimann JM. The evolving concept of a

stem cell: entity or function? Cell 2001; 105:829–41.[2] Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithe-

lium. Surv Ophthalmol 2000; 44:415–25.[3] Hall PA, Watt FM. Stem cells: the generation and maintenance of

cellular diversity. Development 1989; 106:619–33.[4] Lavker RM, Sun TT. Epidermal stem cells: properties, markers, and

location. Proc Natl Acad Sci USA 2000; 97:13473–5.[5] Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls

and uncertainties. Lessons for and from the crypt. Development1990; 110:1001–20.

[6] Potten CS, Morris RJ. Epithelial stem cells in vivo. J Cell Sci Suppl1988; 10:45–62.

[7] Slack JM. Stem cells in epithelial tissues. Science 2000; 287:1431–3.[8] Schofield R. The relationship between the spleen colony-forming

cell and the haemopoietic stem cell. Blood Cells 1978; 4:7–25.[9] Trentin, J. Regulation of hematopoietic stem cells. In: Gordon, A.

(eds). Regulation of hematopoietic stem cells. New York: Appleton-Century-Crofts, 1970; 161–185.

[10] Hanna C, O’Brien JE. Cell production and migration in the epitheliallayer of teh cornea. Arch Ophthalmol 1960; 64:536–9.

[11] Hanna C, O’Brien JE. Cell turnover in the adult human eye. ArchOphthalmol 1961; 65:695–8.

[12] Dua HS, Forrester JV. The corneoscleral limbus in human cornealepithelial wound healing. Am J Ophthalmol 1990; 110:646–56.

[13] Davanger M, Evensen A. Role of the pericorneal papillary structurein renewal of corneal epithelium. Nature 1971; 229:560–1.

[14] Zieske, JD. Perpetuation of stem cells in the eye. Eye 1994; 8(Pt 2):163–9.

[15] Tseng SC. Concept and application of limbal stem cells. Eye 1989;3 (Pt 2):141–57.

[16] Sun, TT, Lavker RM. Corneal epithelial stem cells: past, present, andfuture. J Investig Dermatol Symp Proc 2004; 9:202–7.

[17] Boulton M, Albon J. Stem cells in the eye. Int J Biochem Cell Biol2004; 36:643–57.

[18] Levin LA, Ritch R, Richards JE, Borras T. Stem cell therapy for oculardisorders. Arch Ophthalmol 2004; 122:621–7.

[19] Dua HS, Joseph A, Shanmuganathan VA, Jones RE. Stem cell differ-entiation and the effects of deficiency. Eye 2003; 17:877–85.

[20] Wolosin JM, Xiong X, Schutte M, Stegman Z, Tieng A. Stem cells anddifferentiation stages in the limbo-corneal epithelium. Prog Retin EyeRes 2000; 19:223–55.

[21] Buck RC. Cell migration in repair of mouse corneal epithelium. InvestOphthalmol Vis Sci 1979; 18:767–84.

[22] Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial main-tenance. Invest Ophthalmol Vis Sci 1983; 24:1442–3.

[23] Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalogof human cytokeratins: patterns of expression in normal epithelia,tumors and cultured cells. Cell 1982; 31:11–24.

[24] Tseng SC, Jarvinen MJ, Nelson WG, Huang JW, Woodcock-MitchellJ, Sun TT. Correlation of specific keratins with different types ofepithelial differentiation: monoclonal antibody studies. Cell 1982;30:361–72.

[25] Sun TT, Eichner R, Schermer A, Cooper D, Nelson WG, Weiss RA.Classification, expression, and possible mechanisms of evolution ofmammalian epithelial keratins: A unifying model. In: Levine, A.,Topp, W., Woude, V. G. and Watson, J. D. (eds). Classification, Ex-pression, and Possible Mechanisms of Evolution of Mammalian Ep-ithelial Keratins: A Unifying Model. New York: Cold Spring Harbor,1984; 169–176.

[26] Schermer A., Galvin S, Sun TT. Differentiation-related expression ofa major 64K corneal keratin in vivo and in culture suggests limballocation of corneal epithelial stem cells. J Cell Biol 1986; 103:49–62.

[27] Lavker RM, Sun TT. Epithelial stem cells: the eye provides a vision.Eye 2003; 17:937–42.

[28] Bickenbach JR, Holbrook KA. Label-retaining cells in human embry-onic and fetal epidermis. J Invest Dermatol 1987; 88:42–6.

[29] Bickenbach JR, Mackenzie IC. Identification and localization of label-retaining cells in hamster epithelia. J Invest Dermatol 1984; 82:618–22.

[30] Cotsarelis G, Cheng SZ., Dong G, Sun TT, Lavker RM. Existence ofslow-cycling limbal epithelial basal cells that can be preferentiallystimulated to proliferate: implications on epithelial stem cells. Cell1989; 57:201–9.

[31] Morris RJ, Potten CS. Slowly cycling (label-retaining) epidermal cellsbehave like clonogenic stem cells in vitro. Cell Prolif 1994; 27:279–89.

[32] Lehrer MS, Sun TT, Lavker RM. Strategies of epithelial repair: modu-lation of stem cell and transit amplifying cell proliferation. J Cell Sci1998; 111 (Pt 19):2867–75.

[33] Ebato B, Friend J, Thoft RA. Comparison of limbal and peripheralhuman corneal epithelium in tissue culture. Invest Ophthalmol VisSci 1988; 29:1533–7.

[34] Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, RamaP, De Luca, M. Location and clonal analysis of stem cells and theirdifferentiated progeny in the human ocular surface. J Cell Biol 1999;145:769–82.

[35] Lavker RM., Wei ZG, Sun TT. Phorbol ester preferentially stimulatesmouse fornical conjunctival and limbal epithelial cells to proliferatein vivo. Invest Ophthalmol Vis Sci 1998; 39:301–7.

[36] Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stemcells and their niche. Cell 2004; 116:769–78.

[37] Spangrude GJ., Heimfeld S, Weissman IL. Purification and character-ization of mouse hematopoietic stem cells. Science 1988; 241:58–62.

[38] Blanpain C, Lowry WE., Geoghegan A, Polak L, Fuchs E. Self-renewal, multipotency, and the existence of two cell populationswithin an epithelial stem cell niche. Cell 2004; 118:635–48.

[39] Turksen K. Revisiting the bulge. Dev Cell 2004; 6:454–6.[40] Vogt A. Atlas of Slitlamp Microscopy of the Living Eye. Berlin:

Springer, 1921.[41] Gipson IK. The epithelial basement membrane zone of the limbus.

Eye 1989; 3 (Pt 2):132–40.

P. Charukamnoetkanok 6

Sem

in O

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02/2

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For

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onal

use

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[42] Goldberg MF, Bron AJ. Limbal palisades of Vogt. Trans Am Ophthal-mol Soc 1982; 80:155–71.

[43] Townsend WM. The limbal palisades of Vogt. Trans Am OphthalmolSoc 1991; 89:721–56.

[44] Philipp W, Speicher L, Humpel C. Expression of vascular endothelialgrowth factor and its receptors in inflamed and vascularized humancorneas. Invest Ophthalmol Vis Sci 2000; 41:2514–22.

[45] Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M,Magner M, Isner JM, Asahara T. Ischemia- and cytokine-inducedmobilization of bone marrow-derived endothelial progenitor cellsfor neovascularization. Nat Med 1999; 5:434–8.

[46] Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization.Curr Opin Ophthalmol 2001; 12:242–9.

[47] Colby KA, Adamis AP. Prevalence of corneal neovascularization in ageneral eye service population. Invest Ophthalmol Vis Sci 1996; 37(suppl): S593.

[48] World Health Organization. Methodology for Trachoma Control.WHO Tech Rep Ser 1970; 703.

[49] Lee P, Wang CC, Adamis AP. Ocular neovascularization: an epidemi-ologic review. Surv Ophthalmol 1998; 43:245–69.

[50] Klintworth GK. Corneal Angiogenesis: A Comprehensive Critical Re-view. New York: Springer-Verlag, 1991.

[51] Nishida K, Kinoshita S, Ohashi Y, Kuwayama Y, Yamamoto S. Ocularsurface abnormalities in aniridia. Am J Ophthalmol 1995; 120:368–75.

[52] Kenyon KR, Tseng SC. Limbal autograft transplantation for ocu-lar surface disorders. Ophthalmology 1989; 96:709–22; discussion722–3.

[53] Holland EJ, Schwartz GS. Iatrogenic limbal stem cell deficiency. TransAm Ophthalmol Soc 1997; 95:95–107; discussion 107–10.

[54] Holland EJ, Schwartz GS. Epithelial stem-cell transplantation for se-vere ocular-surface disease. N Engl J Med 1999; 340:1752–3.

[55] Holland EJ, Schwartz GS. Changing concepts in the management ofsevere ocular surface disease over twenty-five years. Cornea 2000;19:688–98.

[56] Djalilian AR., Holland EJ, Schwartz GS. Limbal stem cell deficiency.Ophthalmology 2003; 110:2071; author reply 2071–2.

[57] Holland EJ, Schwartz GS. The evolution of epithelial transplantationfor severe ocular surface disease and a proposed classificationsystem. Cornea 1996; 15:549–56.

[58] Solomon A, Ellies P, Anderson DF, Touhami A, Grueterich M, EspanaEM., Ti SE, Goto E, Feuer WJ, Tseng SC. Long-term outcome of kera-tolimbal allograft with or without penetrating keratoplasty for totallimbal stem cell deficiency. Ophthalmology 2002; 109:1159–66.

[59] Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H, Shimazaki J. Treatment of severe ocular-surfacedisorders with corneal epithelial stem-cell transplantation. N Engl JMed 1999; 340:1697–703.

[60] Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, DeLuca, M. Long-term restoration of damaged corneal surfaces withautologous cultivated corneal epithelium. Lancet 1997; 349:990–3.

[61] Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bio-engineered tissue replacements in patients with ocular surfacedisease. Cornea 2000; 19:421–6.

[62] Tsai RJ, Li L M, Chen JK. Reconstruction of damaged corneas bytransplantation of autologous limbal epithelial cells. N Engl J Med2000; 343:86–93.

[63] Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li DQ.Characterization of putative stem cell phenotype in human limbalepithelia. Stem Cells 2004; 22:355–66.

[64] Chirila TV. An overview of the development of artificial corneaswith porous skirts and the use of PHEMA for such an application.Biomaterials 2001; 22:3311–7.

[65] Khan B, Dudenhoefer EJ, Dohlman CH. Keratoprosthesis: anupdate. Curr Opin Ophthalmol 2001; 12:282–7.

[66] Hicks CR, Fitton JH, Chirila TV, Crawford GJ, Constable IJ. Ker-atoprostheses: advancing toward a true artificial cornea. SurvOphthalmol 1997; 42:175–89.

[67] Taylor DM, Pamel GJ. Keratoprosthesis: possible utilization indeveloping countries. Refract Corneal Surg 1991; 7:472–3.

[68] Barber JC. Keratoprosthesis: past and present. Int Ophthalmol Clin1988; 28:103–9.

[69] Carlsson DJ, Li F, Shimmura S, Griffith M. Bioengineered corneas:how close are we? Curr Opin Ophthalmol 2003; 14:192–7.

[70] Yaghouti F, Dohlman CH. Innovations in keratoprosthesis: provedand unproved. Int Ophthalmol Clin 1999; 39:27–36.

[71] Hicks CR, Crawford GJ, Tan DT, Snibson GR, Sutton GL, DownieN, Gondhowiardjo TD, Lam DS, Werner L, Apple D, Constable IJ. AlphaCor cases: comparative outcomes. Cornea 2003; 22:583–90.

[72] Dohlman CH, Doane MG. Some factors influencing outcome afterkeratoprosthesis surgery. Cornea 1994; 13:214–8.

[73] Yaghouti F, Nouri M, Abad JC, Power WJ, Doane MG, DohlmanCH. Keratoprosthesis: preoperative prognostic categories. Cornea2001; 20:19–23.

[74] Shimazaki J, Shimmura S, Mochizuki K, Tsubota K. Morphologyand barrier function of the corneal epithelium after penetratingkeratoplasty: association with original diseases, tear function, andsuture removal. Cornea 1999; 18:559–64.

[75] Dohlman CH, Dudenhoefer EJ, Khan BF, Morneault S. Protectionof the ocular surface after keratoprosthesis surgery: the role of softcontact lenses. Clao J 2002; 28:72–4.

[76] Netland PA, Terada H, Dohlman CH. Glaucoma associated withkeratoprosthesis. Ophthalmology 1998; 105:751–7.

[77] Nouri M, Terada H, Alfonso EC, Foster CS, Durand ML, DohlmanCH. Endophthalmitis after keratoprosthesis: incidence, bacterialcauses, and risk factors. Arch Ophthalmol 2001; 119:484–9.

[78] Lauweryns B, van den Oord JJ, Missotten L. The transitional zonebetween limbus and peripheral cornea. An immunohistochemicalstudy. Invest Ophthalmol Vis Sci 1993; 34:1991–9.

[79] Lauweryns B, van den Oord JJ, De Vos R, Missotten L. A newepithelial cell type in the human cornea. Invest Ophthalmol Vis Sci1993; 34:1983–90.

[80] Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I,Bondanza S, Ponzin D, McKeon F, De Luca M. p63 identifieskeratinocyte stem cells. Proc Natl Acad Sci USA 2001; 98:3156–61.

[81] de Paiva CS, Chen Z, Corrales RM., Pflugfelder SC, Li DQ. ABCG2transporter identifies a population of clonogenic human limbalepithelial cells. Stem Cells 2005; 23:63–73.

[82] Sarkadi B, Ozvegy-Laczka C, Nemet K, Varadi A. ABCG2—atransporter for all seasons. FEBS Lett 2004; 567:116–20.

[83] Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, MorrisJJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP.The ABC transporter Bcrp1/ABCG2 is expressed in a wide varietyof stem cells and is a molecular determinant of the side-populationphenotype. Nat Med 2001; 7:1028–34.

[84] Lauweryns B, van den Oord JJ, Volpes R, Foets B, Missotten L.Distribution of very late activation integrins in the human cornea.An immunohistochemical study using monoclonal antibodies.Invest Ophthalmol Vis Sci 1991; 32:2079–85.

[85] Pancholi V. Multifunctional alpha-enolase: its role in diseases. CellMol Life Sci 2001; 58:902–20.

[86] Zieske JD, Bukusoglu G, Yankauckas, M. A. Characterization of apotential marker of corneal epithelial stem cells. Invest OphthalmolVis Sci 1992; 33:143–52.

[87] Zieske JD, Wasson M. Regional variation in distribution of EGFreceptor in developing and adult corneal epithelium. J Cell Sci 1993;106 (Pt 1):145–52.

[88] Wolosin JM, Schutte M, Zieske JD, Budak MT. Changes in con-nexin43 in early ocular surface development. Curr Eye Res 2002;24:430–8.

[89] Scott RA, Lauweryns B, Snead DM, Haynes RJ, Mahida Y, DuaHS. E-cadherin distribution and epithelial basement membranecharacteristics of the normal human conjunctiva and cornea. Eye1997; 11 (Pt 5):607–12.

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