extended report correlation of long term phenotypic and … · technique. to ascertain safety and...

EXTENDED REPORT

Correlation of long term phenotypic and clinical outcomesfollowing limbal epithelial transplantation cultivated onamniotic membrane in rabbitsS-E Ti, M Grueterich, E M Espana, A Touhami, D F Anderson, S C G Tseng. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Scheffer C G Tseng, MD,PhD, Ocular SurfaceCenter, 7000 SW 97thAvenue, Suite 213, Miami,FL 33173, USA;[email protected]

Accepted for publication1 August 2003. . . . . . . . . . . . . . . . . . . . . . .

Br J Ophthalmol 2004;88:422–427. doi: 10.1136/bjo.2003.026054

Aim: To determine the epithelial phenotype in rabbits with total limbal stem cell deficiency (LSCD) afterreconstruction with autologous limbal epithelial stem cells ex vivo expanded on rabbit amniotic membrane(AM).Methods: Left eyes of 52 rabbits were rendered total LSCD, verified by impression cytology. Thefibrovascular pannus of each cornea was removed. Group I (n = 10) received rabbit AM transplantationalone, while groups II–IV (n = 42) underwent transplantation of LSC cultured on rabbit AM (LSC-AM) froma small limbal biopsy taken from the right eye. Clinical outcome was graded as ‘‘success,’’ ‘‘partialsuccess,’’ or ‘‘failure’’ depending on the corneal smoothness and avascularity. Epithelial phenotype wasdetermined by immunostaining and graded as ‘‘corneal (K),’’ ‘‘conjunctival (J),’’ or ‘‘mixed (M)’’depending on expression of K3 and Muc5AC.Results: After 1 year follow up, group I showed 100% failure and groups II–IV showed 26% success(p,0.001). Clinical failure correlated with J phenotype p = 0.001), while clinical success correlated with Kphenotype p = 0.01). When the phenotypic outcome was used for comparison, J phenotype wassignificantly high in group I (p = 0.003), while K phenotype was significantly high in groups II–IV (p,0.05).Conclusion: There is a strong correlation between clinical success and resultant corneal epithelialphenotype. Ex vivo expanded LSC can successfully reconstruct corneal surfaces with unilateral total LSCD.

The integrity of the corneal epithelium depends on itsepithelial stem cells located at the limbus. Limbalepithelial stem cells (LSC) serve as the ultimate source

for maintenance and regeneration of the corneal epithe-lium.1–3 Damage or dysfunction of the limbus results in astate termed limbal stem cell deficiency (LSCD), which canbe found by impression cytology in a number of ocularsurface diseases.4 Restoration of corneal surfaces in patientswith LSCD resorts to transplantation of autologous orallogeneic LSC.5 6

To avoid potential complications to the living donor eye intraditional autologous and allogeneic limbal transplantation,a new surgical procedure has successfully been practised inhuman patients with partial or total LSCD by expanding LSCex vivo in culture from a small limbal biopsy.7–11

Undoubtedly, these human clinical experiences lend signifi-cant insight to our understanding of this cultivationtechnique.

To ascertain safety and clinical efficacy of this new surgicalprocedure, we have used an animal model to study culturingand surgical variables affecting the long term outcomefollowing transplantation of ex vivo expanded LSC.12 Wehave identified that a single most important factor promotingclinical success is to apply an additional amniotic membrane(AM) as a temporary patch over the composite graftconsisting of cultivated LSC on AM so that epithelialisationcan be ensured at the early stage.12 After 1 year follow up, weverified that clinical outcome of success is correlated with therecovery of a normal corneal epithelial phenotype. Thesignificance of these findings is further discussed.

MATERIALS AND METHODSMaterialsMonoclonal antibodies against keratin K3 (AE-5 1:100) werefrom ICN (Aurora, OH, USA) and goblet cell mucin (MUC

5AC 1:100) was a kind gift from Jacques Bara, MD, Paris,France. Other materials were obtained as reported.12

AnimalsA total of 65 New Zealand White rabbits (divided into fourstudy groups were included in this study protocol approvedby the animal research committee of the University of Miami(No 01–127) and followed guidelines described in the ARVOstatement for the use of animals in ophthalmic and visionresearch. After excluding 13 rabbits because of surgicalcomplications, the remaining 52 rabbits were subdivided intofour groups.

Creation of total LSCD modelThe model of total LSCD was created in the left eye of allrabbits using a method previously described.13 14 The ocularsurface became progressively conjunctivalised, with ingrow-ing fibrovascular pannus. Impression cytology was performedto confirm total LSCD using a previous method15 and basedon a published diagnostic criterion.4

Preparation of rabbit AMRabbit AM was harvested from 28 day pregnant does understerile conditions, prepared and preserved as previouslydescribed.16

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Abbreviations: AM, amniotic membrane; AMT, amniotic membranetransplantation; LSC, limbal epithelial stem cells; LSCD, limbal stem celldeficiency

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Preparation of AM inserts, l imbal biopsy, and cultureon AMAfter thawing and rinsing in HBSS, the AM was fastenedonto inserts.17 A limbal biopsy was performed in the normalright eye of each rabbit in groups II and III. The method of exvivo expansion from the limbal biopsy specimen has beendescribed.12

Surgical transplantationThe left eye of each rabbit in all groups underwent 360˚conjunctival peritomy followed by removal of the fibrovas-cular pannus.18 Group I (n = 10) underwent rabbit AMtransplantation (AMT) alone as previously described.19

Groups II–IV (n = 42) underwent AMT with ex vivoexpanded limbal epithelial cells and differences among themwere previously reported.12

Definit ion of clinical successWhenever the cornea became fully vascularised—that is,reverting to a preoperative appearance, the animal was killedand rated as a failure. Otherwise, all animals were followedup for more than 1 year before sacrifice. If the cornearegained its smoothness, avascularity, and clarity the animalwas graded as a success. If the cornea showed a mixture ofthe above two features—that is, two quadrants or more ofclear cornea, the eye was graded as a partial success.

Phenotypic analysisOutcome was also measured by the epithelial phenotype ofthe transplanted corneas obtained after sacrifice by haema-toxylin and eosin and periodic acid Schiff (PAS) staining andby immunofluorescence staining with the monoclonal anti-body AE5 to K3 keratin1 and with the monoclonal antibodyAM3 to MUC5AC goblet cell mucin.18–20 Complete success wasdefined if the corneal epithelial phenotype was restored, as

evidenced by the restoration of a stratified epitheliumwithout goblet cell, which was positive to AE5 but negativeto AM3 staining. Failure was defined if the resultantepithelium was of a conjunctival origin, as shown by thepresence of goblet cells, which was negative to AE5 butpositive to AM3 staining. In some eyes, a mixed phenotypewas noted with the resultant epithelium showing a mixtureof the above two phenotypes.

Statistical analysisThe comparison of the frequency of clinical outcomes ofsuccess, partial success, and failure or age and that ofphenotypic outcomes of K, J, or M was performed by Fisher’sexact test. The correlation of clinical outcomes withphenotypic outcomes was performed by two tailed x2 testusing Graph Pad InStat Version 3.05 (Graph Pad Software,Inc, CA, USA). A p value of ,0.05 was considered asstatistically significant.

RESULTSClinical outcomeThe full clinical results have previously been reported.12 Thecumulative percentage survival at 30 and 180 days was 10%and 0% for group 1, 64% and 21% for group II, 77% and 18%for group III, and 100% and 74% for group IV, respectively.The log rank test showed that group IV had statisticallylonger times to transplant failure (that is, significantly highertransplant success) than the other three surgical groups (allp,0.002). The success rate was 0% in group I but 26% ingroups II–IV (p ,0.0001).

Phenotypic outcomeFor groups II, III, and IV, corneas with clinical success wererestored with a smooth and avascular surface and clearstroma (fig 1A–C). Haematoxylin and eosin staining demon-

Figure 1 Correlation of clinicalsuccess with a corneal phenotype.preoperative appearance (A). Aftertransplantation of ex vivo expandedLSC (B). Postoperative appearance (C).Haematoxylin and eosin staining (Dand E). Immunostaining to K3 keratin(F) the entire corneal epithelium in inset,arrow from the limbal region).Immunostaining to MUC5AC in theconjunctival epithelium (G, arrowmarks the limbus).

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strated the recovery of a normal stratified corneal epithelium,a PAS positive new basement membrane, and an avascularstroma with uniform cellularity (fig 1D, E). In some corneas,the amniotic basement membrane was preserved in thetransplanted corneas as a thick eosinophilic layer (fig 1D,bracket). Immunostaining to K3 keratin showed full thick-ness positivity in the entire corneal epithelium (inset, fig 1F)and suprabasal positivity in the limbal region (arrow, fig 1F),while immunostaining to MUC5AC showed conjunctivalgoblet cells only in the conjunctival epithelium (fig 1G).This pattern is identical to the normal corneal epithelialphenotype in vivo and denoted as ‘‘corneal (K) phenotype.’’

In groups II, III, and IV, some corneas with clinical partialsuccess had a smooth and avascular surface and clear stromain most of the cornea (fig 2A–C), while part of the cornea wascovered with vascularised pannus (fig 2C). Haematoxylin andeosin staining showed that the pannus region was covered byan attenuated epithelium and the stroma was infiltrated withchronic inflammatory cells and blood vessels (fig 2D, E).Immunostaining revealed that the majority of the cornealsurface had a K phenotype (fig 2F, G), while the pannus

region was devoid of K3 expression (fig 2F). The pannus wasalso negative to MUC5AC staining (not shown), andconjunctival goblet cells were found only in the conjunctiva(not shown). Therefore, we still graded these corneas as acorneal (K) phenotype (table 1). In contrast, most corneaswith clinical partial success actually showed a mixture ofcorneal and conjunctival phenotypes on the corneal surface.One such example is shown in figure 3, where a stratifiedepithelium was found in one part of the cornea (fig 3D, E),while the other part covered by vascularised pannus,contained a thinner epithelium with goblet cells underhaematoxylin and eosin staining. This mixed phenotype(denoted as M) was verified by the presence of both fullthickness K3 keratin (fig 3F, G) and MUC5AC positive gobletcells (fig 3H) on the corneal surface.

For all groups, corneas with clinical failure continued to becovered by vascularisation and an irregular epithelium(fig 4A, B). Among them, a small portion of corneas showedM phenotype as defined in figure 3, but the majority of themshowed a conjunctival (J) phenotype. Under haematoxylinand eosin staining, the epithelium was not as organised and

Figure 2 Correlation of clinical partialsuccess with a corneal phenotype andgranuloma. In this representative case,the preoperative and postoperativeappearance (A–C). Haematoxylin andeosin staining showed chronicinflammatory cells and blood vessels inpannus (D and E). Immunostaining toK3 keratin was negative in the pannusregion (F, G).

Table 1 Correlation between clinical and phenotypic outcomes

Rabbit

Group I Group II Group III Group IV

Clinical Phenotype Clinical Phenotype Clinical Phenotype Clinical Phenotype

1 F J F M F J PS K2 F J PS J F M S K3 F J F J F J PS M4 F M S K F J S K5 F J F J F J S K6 F J F M PS M S K7 F J S K S K PS K8 F J F M F J S K9 F J F M F M PS M10 F J F M F J PS K11 F J F J S K12 F M F J F J13 F J F J14 F J PS K15 S M S K

F = failure; PS = partial success; S = success; J, = conjunctival phenotype; K = corneal phenotype; M = mixed corneal and conjunctival phenotype.

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stratified, the basement membrane was indistinct, and thestroma was infiltrated with blood vessels and chronicinflammatory cells (fig 4C). Immunostaining showed nega-tive K3 keratin staining (fig 4D), but positive MUC5ACstained goblet cells throughout (fig 4E).

Correlation of clinical and phenotypic outcomesTable 1 summarises clinical and phenotypic outcomes of allfour groups. None of group I (AMT alone, n = 10) had clinicalsuccess. In groups II, III, and IV (n = 42), there were a total of11 clinical successes (p,0.0001). Except for one, which had a

conjunctival phenotype, the rest 10 had a corneal phenotype.None of group I had partial clinical success either. However,in groups II, III, and IV, there were a total of eight clinicalpartial successes, of which four corneas had a cornealphenotype, three had a mixed phenotype, and one had aconjunctival phenotype. All 10 eyes in group I showed clinicalfailures. Except for one eye, which had a mixed phenotype,the rest (nine) had a conjunctival phenotype. In groups II, III,and IV, there were a total of 23 clinical failures, of which 15eyes had a conjunctival phenotype, and eight eyes had amixed phenotype. Clinical failure correlated with a conjunc-

Figure 3 Correlation of clinical partialsuccess with a mixed phenotype. In thisrepresentative case, preoperative andpostoperative appearance (A–C).Haematoxylin and eosin staining (Dand E). Immunostaining to K3 keratin (Fand G), and MUC5AC positive gobletcells (H) on the corneal surface.

Figure 4 Correlation of clinical failurewith a conjunctival phenotype. In thisrepresentative case, preoperative andpostoperative appearances (A, B)haematoxylin and eosin staining (C,arrow marks goblet cells).Immunostaining showed negative K3keratin staining (D), but positiveMUC5AC stained goblet cellsthroughout (E).





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tival phenotype (p,0.001), while clinical success correlatedwith a corneal phenotype (p,0.01). Clinical partial successcould be either a corneal phenotype or a mixed phenotype(p,0.05). When the phenotypic outcome was used to assessthe efficacy of this procedure, we noted that the conjunctivalphenotype was significantly more in group I (p,0.003),while the corneal phenotype was significantly more in groupsII–IV (p,0.05), especially in group IV.

DISCUSSIONPreviously we reported that autologous limbal epithelial cellsexpanded ex vivo on amniotic membrane is effective inrestoring a normal corneal surface in this rabbit model oftotal LSCD.12 In this study, we provided additional evidenceto support that such clinical successes correlated well withthe restoration of a non-keratinised stratified epitheliumwithout goblet cells (fig 1D and E), and a recovery of acorneal epithelial phenotype based on the expression ofcorneal epithelium specific K3 keratin (fig 1F) and the lack ofexpression of conjunctival goblet cell specific MUC5AC(fig 1G). As reported,12 clinical success was not found inany of 10 eyes (0%) of group I with amniotic membranealone, but was found in 11 of 42 eyes (26%) in groups II, III,and IV (p,0.0001) with additional ex vivo expanded limbalepithelial cells. Among these 11 eyes, 10 (91%) had a cornealepithelial phenotype, confirming that ex vivo expandedlimbal epithelial cells were responsible for the restoration ofa normal corneal epithelial phenotype. This finding isconsistent with our laboratory data showing that amnioticmembrane cultures help expand limbal epithelial SC.21–23

Because such a clinical success and a corneal phenotypelasted for more than 1 year after transplantation, we furtherconfirmed that ex vivo expansion of limbal epithelium is avalid procedure to treat unilateral total LSCD. Because asignificantly higher percentage of clinical success and cornealphenotype was noted in group IV, we also believe protectionof newly transplanted limbal epithelium by another amnioticmembrane is crucial to ensure this success.

In contrast, clinical failures correlated well with aconjunctival epithelial phenotype, verified by the lack of K3keratin expression (fig 4D) but positive expression ofMUC5AC (fig 4E). As reported,12 clinical failures were notedin all 10 eyes of group I, of which all except one had aconjunctival epithelial phenotype. Clinical failures were alsonoted in 23 of 42 eyes of groups II, III, and IV. We noted thatclinical failure correlated with a conjunctival epithelialphenotype (p = 0.001). Among these 23 eyes, 15 eyes (65%)had a conjunctival phenotype, while the rest (eight) eyes(35%) had a mixed phenotype. This result indicated thatsome clinical failures might not be associated with acomplete loss of the corneal epithelial phenotype. Althoughwe cannot resolve whether these residual corneal epithelialcells represent a smaller portion of limbal epithelial SC orsimply remaining corneal epithelial TAC (transient amplify-ing cells), we did learn from our earlier report12 that thesefailures correlate well with the early epithelial defects in thefirst 3 months after transplantation because of exposureproblems in groups II and III.

In groups II, III, and IV, four of eight eyes (50%) showed acorneal epithelial phenotype (fig 2), suggesting that thepannus noted clinically was not caused by ‘‘conjunctivalisa-tion’’ but rather by ‘‘granuloma.’’ Therefore, judging from thephenotypic outcome, the success rate was further elevated to75%. Three of eight eyes (38%) showed a mixed phenotype(fig 3), suggesting that the pannus noted in these eyes wascaused by focal conjunctivalisation—that is, partial LSCD.Differentiation of partial LSCD from granuloma by impres-sion cytology is clinically important to guide effectivetherapies.

These end point outcome analyses did not reveal thebiological process during the wound healing. For example,successful corneas showed progressive wound remodell-ing from an opaque amniotic membrane to transparentcorneal stroma. We thus speculate that transplantation ofcorneal epithelial cells aids the return of stromal clarity.Further studies of such wound healing processes leadingto the restoration of corneal stroma will help us under-stand how scarless wound healing may be achieved in thecornea.

ACKNOWLEDGEMENTSSupported by Public Health Service Research Grant EY06819 (toSCGT via TissueTech, Inc) from Department of Health and HumanServices, National Eye Institute, National Institute of Health,Bethesda, Maryland, Human Manpower Development Program,Singapore National Eye Centre, Singapore (to SET), ResearchFellowship grant GR-1814/1–2 (to MG) by the DeutscheForschungsgemeinschaft, Bonn, Germany, and a grant from OcularSurface Research and Education Foundation.

Authors’ affiliations. . . . . . . . . . . . . . . . . . . . .

S-E Ti, M Grueterich, E M Espana, A Touhami, S C G Tseng, TissueTech,Inc, and Ocular Surface Center, Miami, FL, USAM Grueterich, D F Anderson, S C G Tseng, Bascom Palmer Eye Institute,University of Miami School of Medicine, Miami, FL, USAS-E Ti, Singapore National Eye Centre, Singapore

Proprietary interest: SCGT and his family are more than 5% shareholdersof TissueTech, Inc, which owns US patents Nos 6,152,142 and6,326,019 on the method of preparation and clinical uses of humanamniotic membrane distributed by Bio-Tissue, Inc.

Presented in part at the annual meeting of ARVO in Ft Lauderdale, FL,USA, in May 2003

REFERENCES1 Schermer A, Galvin S, Sun T-T. Differentiation-related expression of a major

64K corneal keratin in vivo and in culture suggests limbal location of cornealepithelial stem cells. J Cell Biol 1986;103:49–62.

2 Cotsarelis G, Cheng SZ, Dong G, et al. Existence of slow-cycling limbalepithelial basal cells that can be preferentially stimulated to proliferate:implications on epithelial stem cells. Cell 1989;57:201–9.

3 Tseng SCG. Regulation and clinical implications of corneal epithelial stemcells. Mol Biol Rep 1996;23:47–58.

4 Puangsricharern V, Tseng SCG. Cytologic evidence of corneal diseases withlimbal stem cell deficiency. Ophthalmology 1995;102:1476–85.

5 Tseng SCG. Conjunctival grafting for corneal diseases. In: Tasman W,Jaeger EA, eds. Duane’s clinical ophthalmology. Philadelphia: JB Lippincott,1994:1–11.

6 Holland EJ, Schwartz GS. The evolution of epithelial transplantation for severeocular surface disease and a proposed classification system. Cornea1996;15:549–56.

7 Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damagedcorneal surface with autologous cultivated corneal epithelium. Lancet1997;349:990–3.

8 Tsai RJF, Li L-M, Chen J-K. Reconstruction of damaged corneas bytransplantation of autologous limbal epithelial cells. N Engl J Med2000;343:86–93.

9 Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelialtransplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119:298–300.

10 Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem celltransplantation in ocular surface disorders. Ophthalmology2001;108:1569–74.

11 Grueterich M, Espana EM, Touhami A, et al. Phenotypic study of a case withsuccessful transplantation of ex vivo expanded human limbal epithelium forunilateral total limbal stem cell deficiency. Ophthalmology2002;109:1547–52.

12 Ti S-E, Anderson DF, Touhami A, et al. Factors affecting outcome followingtransplantation of ex vivo expanded limbal epithelium on amniotic membranefor total limbal deficiency in rabbits. Invest Ophthalmol Vis Sci2002;43:2584–92.

13 Kruse FE, Chen JJY, Tsai RJF, et al. Conjunctival transdifferentiation is due tothe incomplete removal of limbal basal epithelium. Invest Ophthalmol Vis Sci1990;31:1903–13.

14 Huang AJW, Tseng SCG. Corneal epithelial wound healing in the absence oflimbal epithelium. Invest Ophthalmol Vis Sci 1991;32:96–105.

15 Tseng SCG. Staging of conjunctival squamous metaplasia by impressioncytology. Ophthalmology 1985;92:728–33.

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16 Lee S-H, Tseng SCG. Amniotic membrane transplantation for persistentepithelial defects with ulceration. Am J Ophthalmol 1997;123:303–12.

17 Meller D, Tseng SCG. Conjunctival epithelial cell differentiation on amnioticmembrane. Invest Ophthalmol Vis Sci 1999;40:878–86.

18 Tsai RJF, Sun T-T, Tseng SCG. Comparison of limbal and conjunctivalautograft transplantation for corneal surface reconstruction in rabbits.Ophthalmology 1990;97:446–55.

19 Kim JC, Tseng SCG. Transplantation of preserved human amniotic membranefor surface reconstruction in severely damaged rabbit corneas. Cornea1995;14:473–84.

20 Chen JJY, Tseng SCG. Corneal epithelial wound healing in partial limbaldeficiency. Invest Ophthalmol Vis Sci 1990;31:1301–14.

21 Meller D, Pires RTF, Tseng SCG. Ex vivo preservation and expansion of humanlimbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol2002;86:463–71.

22 Grueterich M, Tseng SCG. Human limbal progenitor cells expanded on intactamniotic membrane. Arch Ophthalmol 2003 (in press).

23 Grueterich M, Espana E, Tseng SC. Connexin 43 expression and proliferationof human limbal epithelium on intact and denuded amniotic membrane. InvestOphthalmol Vis Sci 2002;43:63–71.

It says right here, George, ‘‘Individual results may vary.’’ EMichael Balis





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