Ex Vivo Expansion of Human Limbal Stem Cells for Corneal Surface Reconstruction

 

Purpose: To develop a technique to culture the limbal stem cells for clinical corneal surface reconstruction, Methods: In order to engineer a reproducible corneal epithelium which can maintains the proliferative and differentiated functions of the tissue, we have to develop an ex vivo environment for limbal stem cells. The engineer corneal epithelium has to be surgical handling and maintain easily by clinician. We took specimens of limbal epithelial cells from the healthy contralateral eyes of patients with severe unilateral cornea disease or from the donor limbal cornea rim. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane together with the limbal epithelial cell sheet was transplanted to the denuded corneal surface at the disordered eye after superficial keratectomy to remove fibrovascular ingrowth. Results: In 2 to 3 weeks, the limbal epithelial cells grew to form a sheet approximately 2 ×3 cm² in size on the amniotic membrane. On histological examination the epithelial sheet was composed of 4 to 5 cell layers at the margin of the sheet and from 1 to 4 cell layers in the area between the margin and original explant tissue. Ultrastructural examination revealed the presence of intercellular spaces and basement membrane structure with focal condensation of electron-dense ground substance at the basal cell –amniotic membrane junction. After transplantation, there was complete re-epithelization of the corneal surface in 2 to 4 days. By one month, the ocular surface was covered with stable corneal epithelium with improved clarity. The clinical results of auto- and allologous origin of limbal stem cells will be presented. Conclusion: Transplantation of limbal epithelial cells cultured on amniotic membrane provides a simple and effective method to reconstruct the corneal surface and restore useful vision in patients with limbal epithelial cell deficiency, indication limbal stem cells can be preserved. Rejection in allologous origin of limbal stem cells transplantation still is the big problem in outcome.

 

 

 

Title: Proliferating potential of limbal stem cells from cadaveric and live

Donors

 

Presenter: Virender S. Sangwan, MS

 

Associates: Vemuganti GK, Kayshap S, Singh S

 

Affiliation: L.V.Prasad Eye Institute and CCMB, Hyderabad, India

 

Purpose: To evaluate and compare the growth potential of limbal stem cellsfrom cadaveric and live donors. Methods: After obtaining approval of Institution Review Board and informedconsent from the patients, 2x2mm of limbal biopsies were obtained from patients undergoing cataract surgery with no ocular surface or intraocular disease. In similar fashion limbal tissue was harvested from corneoscleral rim after optical penetrating keratoplasty. The tissue was stored either inMK medium or optisol. The biopsies were explanted on the de-epithelized human amniotic membrane and fed with human forneal epithelial cell medium. Growth was observed under phase contrast microscopy for 2 weeks. Whole mount preparation, histochemistry and histologic evaluation were done for confirmation of growth of limbal stem cells.Results: Good growth with a radius of 6-10 mm was observed in92%(12/13) of live tissues, 25%(2/8) of MK medium preserved tissues and 46%(6/13) of Optisol preserved tissues. Conclusion: Fresh tissues from living subjects are a better source of limbal stem cells. Improved techniques of preserving eye bank tissues could enhance the proliferating capacity of stem cells.

 

Key Issue: For clinical limbal allotransplantation cadaveric tissue is poor

source of limbal stem cells and living source is better.

 

 

 

THE EFFECTS OF HEAT SHOCK PROTEINS OF AMNIOTIC MEMBRANE ON PRK

W.C. Park¹, X. Zhou² , D.W. Park¹, H.B Ahn¹, S.H. Rho¹, J.C. Kim³

¹Department of Ophthalmology, Dong-A University, Pusan, Korea. ³Department of Ophthalmology, School of Medicine, Chung-Ang University, Seoul, Korea. ²Department of Ophthalmology, Shanghai Medical University, China.

 

 Purpose : To investigate the role of heat shock proteins (HSPs) on amniotic membrane (AM) using PRK wound healing model.Methods HSPs-rich AM was made by 4⁰C incubation and HSPs-poor AM was made by 37⁰C incubation with fresh AM for 4 days. We checked the amount of HSPs expression in the AM by western blot and immunohistochemical stain for HSP 90, 70, 60, 47.PRK (5mm, -9D) was performed on both eyes of 26 albino rabbits. Immediate after PRK, group A (16 eyes) was grafted with HSPs-rich AM, Group B (16 eyes) was grafted with HSPs-poor AM, Group C (4 eyes) was treated with HSP90 eye drop, 8 times for 48 hours, and Group D (12 eyes) was PRK only as control. Corneal haze was scored for 6 weeks. The corneas were harvested in 24 hours and 6 weeks postoperatively. TUNEL for apoptosis, and H&E stain for PMN infiltration were performed at 24 hours. Collagen II, fibronectin and alpha-SM actin immunohistochemical staining were performed at 6 weeks postoperatively. Results

Group A, B and C showed less TUNEL positive keratocyte, PMN, Collagen II and alpha-AM actin stain than control.group. Group A showed less TUNEL positive keratocyte than group B significantly, but not significance of PMN, Collagen II and alpha-AM actin stain. All group had no difference on cornea opacity. Conlusion Role of HSPs on AM is cytoprotection for keratocytes against damage from excimer laser PRK.

 

 

 

The effect of  amniotic membrane transplantation on limbal and corneal epithelial cell proliferation during corneal wound healing

 

T. H. Choi M.D., J. Y. Lee M.D., D. H. Chun M.D.

Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul Korea

 

Purpose: To evaluate the effect of amniotic membrane transplantation on corneal epithelial wound healing in limbal and peripheral cornea. Method: Amniotic membrane was transplanted to cover the 6mm de-epithelialized  central cornea of  6 rabbit eyes and T-lens was applied to another 6 rabbit eyes as a control. All animals were injected with 10 mM BrdU into the lateral ear vein immediately after surgery and  euthanized at 12, 24, or 48 hrs postoperatively. Immunofluorescent staining with anti-BrdU monoclonal Ab and immunohistochemical staining for PCNA and keratinocyte growth factor (KGF) were performed. BrdU-positive cells and labeling indices for PCNA were assessed for both limbal and peripheral epithelial cells. Results: BrdU and PCNA labeling was greatest at 24 hr in both AM and control groups, but AM group showed higher expression than the control as early as 12 hr postoperatively (p<0.05). Limbal fibroblasts of AM group expressed more KGF than the control. Conclusion: Amniotic membrane enhanced wound healing by stimulating limbal epithelial cell proliferation which is indirectly mediated in part by upregulating the expression of KGF, which is a potent epithelial mitogen secreted by limbal fibroblasts.

 

 

 

tHE amniotic membrane transplantation IN Experimentally alkali burned rabbit cornea

 

Purpose. The purpose of this study is to see the effects of amniotic membrane transplantation on corneal epithelial & stromal wound healing after central corneal alkali wound and its implications on the corneal stromal wound healing. Methods A 5.5 mm round filter paper soaked in 1 N NaOH was applied on one eye of 30 New Zealand white rabbits for 60sec. Human amniotic membrane transplantation(AMT) was performed in 18 eyes immediately after alkali wound. 12 eyes without AMT were used as control. We evaluated epithelial healing patterns and the densities of corneal keratocytes and PMNs in the excised corneas after  24 hours, 48 hours, 3 days, 5 days, 1 week, and 3 weeks. We also evaluated the proliferating cells using monoclonal antibody to proliferating cell nuclear antigen (PCNA). Results. The healing pattern of corneal epithelia & the density of the keratocytes were the same in two groups but the densities of PMNs in AMT group were markedly decreased throughout the period. The density of the proliferating keratocytes were the same in both groups. Conclusions. Amniotic membrane transplantation prevents inflammatory cellular infiltration in alkali-burned rabbit corneas, but has less effect either on the epithelial healing pattern or the density and the proliferation of the keratocytes in alkali-burned rabbit corneas.

 

 

 

Purpose: To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cell sheets on amniotic membrane (AM) for use in ocular surface reconstruction. Methods: Small pieces of corneal limbal epithelium obtained from donor corneas were cultivated for 4 weeks on acellular AM with inactivated 3T3 fibroblasts. At the fourth week, they were divided into 2 groups. The air-lift (-) group was continuously submerged in SHEM medium for the entire 4 weeks. The air-lift (+) group was submerged in medium for 3 weeks, then exposed to air by lowering the medium level (air-lifting) for 1 week. Cultured corneal epithelium morphology and permeability to horseradish peroxidase (HRP) were examined by histochemical staining and transmission electron microscopy. Changes in tight junction-related protein expression were assessed by immunoblotting. Reverse transcription-polymerase chain reaction was used to reveal the expression of mRNA for occludin and various claudins Results: The cultures of both groups formed 4-5 layer-thick, well stratified epithelium. In the air-lifting (+) group, the epithelial cells were tightly attached to each other and the HRP/diaminobenzidine (DAB) reaction product all accumulated on the apical surface of the superficial cells. In the air-lifting (-) group, by contrast, the epithelial cells loosely attached to each other and bigger intercellular spaces were detected. The HRP/DAB reaction product penetrated into the intercellular space to a depth of 3 to 4 cell layers. With the air-lifting technique, tight junction-related proteins localized at the apical portion of the lateral membrane. Tight junction-related proteins and mRNA did not change by air-lifting. Claudin subtype expression became more constant and more similar to that of in vivo corneal epithelium. Conclusions: The air-lifting technique helps make cell-cell attachment tighter and helps create the superficial epithelial cell barrier. This technique is useful in culturing corneal epithelial cells for ocular surface reconstruction. The Key Issue is: Tight junction, cornea epithelial cell, amniotic membrane, ocular surface reconstruction

 

 

 

Title: Human corneal epithelial cells cultured by air-lifting on human

amniotic membrane

 

Presenter: Yuriko Ban 1

 

Associates: Takahiro Nakamura2, Masakastu Tsuzuki2, Atsuyoshi Data2, Noriko

Koizumi2, Nigel J. Fullwood3, Leanne Cooper3, Chikako Mochida2, Shigeru

 

Kinoshita2

Affiliation: Nantan General Hospital, Kyoto, Japan1; Dept. of Ophthalmol.,

Kyoto Prefec. Univ. of Med., Kyoto, Japan 2; Insti. of Environmental and

Natural Sci., Lancaster Univ., U.K. 3

 

Clinical efficacy of ex vivo expansion of limbal epithelial cells for treating limbal deficiency has been reported. We evaluated several surgical variables in a rabbit model. Based on clinical photography, transplantation of rabbit amniotic membrane (AM) resulted in 0% success (n=9). In a total of 30 rabbits (n=8 with interrupted sutures, n=7 with continuous sutures, n=9 with continuous sutures and tarsorrhaphy, and n=6 with tarsorrhaphy and subconjunctival injection of long-acting steroid), we noted an overall clinical success rate of 13% (4/30, once success in each of the four subgroups). This low success rate was found to correlate with the an early occurrence of epithelial defect (within one week), suggesting that the transplanted epithelial cell layer had a poor adhesion despite the protection by tarsorrhphy. This hypothesis was further confirmed by adding another layer of human AM over the transplanted epithelium during surgery in another 8 rabbits. Our recent result showed that there was no early epithelial defect, and a clinical success rate of 6/8 (75%) and partial success of 2/8 (25%). We will also report our data of histologic analysis and phenotypic studies using immunostaining. 

 

 

 

Title:  Examination of Surgical Variables Important to Improve Outcomes of Ex Vivo

Expansion of Limbal Epithelial Stem Cells for Treating Rabbit Unilateral Total

Limbal Stem Cell Deficiency

 

Presenter:               Scheffer C. G. Tseng

 

The Key Issue is: It is possible to repeat the cultivated corneal epithelial transplantation.

 

 

 

Title: Regrafting of cultivated corneal epithelial transplantation in severe ocular surface disease

Presenter: Takahiro Nakamura

 

Associates: N. Koizumi, M. Tsuzuki, Y. Sano, C. Sotozono, and S. Kinoshita

 

Affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. Tel: +81-75-251-5578 Fax: +81-75-251-5663

E-mail: tnakamur@ophth.kpu-m.ac.jp

Abstract Form

Introduction: Our group has performed allo cultivated corneal epithelial transplantation on 23 eyes with acute and chronic phase severe ocular surface disorders. After clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients and achieved successfulocularsurfacereconstruction.Materials and Methods: The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane(AM) carrier, with 3T3 fibroblast co-culture and air-lifting with minor modification of cell preparation. The study subjects consisted of 4 patients (SJS, drug-induced pseudo-pemphigoid and chemical burn). At 3 months and 12 months after the first operation, the failed epithelial graft with AM was replaced again with new allo-corneal epithelium cultivated on AM.Results: At 48 hours after transplantation, the corneal surfaces of the 4 eyes were clear and smooth, and the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable, without epithelial defects. Conclusions: We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.

 

 

 

Title: ELECTRON MICROSCOPIC INVESTIGATION INTO THE REMOVED
CULTIVATED EPITHELIAL TRANSPLANTS IN PATIENTS AFTER ALLO-
CULTIVATED CORNEAL EPITHELIAL TRANSPLAN

 

Presenter:Dr NigelFullwood

Associates:CooperLJBsc.,1KoizumiNM.D.,Ph.D.,2
NakamuraTM.D.,KinoshitaSM.D.,Ph.D.2
 
Affiliation:1BiologicalSciences,LancasterUniversity,U.K;
 2 Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan

 

We investigated the ultrastructural changes of removed cultivated corneal epithelium transplants using scanning electron and transmission electron microscopy. The transplants had been removed due to graft opacities from three patients who had undergone allo-cultivated corneal epithelial transplantation. Their primary diagnoses consisted of one with acute phase chemical burn, one with drug-induced pseudopemphigoid and one with Stevens-Johnson syndrome. Cultivated corneal limbal epithelium using an amniotic membrane carrier was transplanted as previously described (Koizumi et al 2001) and the transplants were removed after several months due to graft opacities after allograft rejection in two cases and after bacterial infection in one case. The removed transplants were prepared for examination by electron microscopy. In all three cases the amniotic membrane remained intact, although it had become partially vascularised and invaded by scleral keratocytes. Inflammatory cells were present in the epithelial layer and within the amniotic membrane. Most of the amniotic membrane was covered by conjunctival epithelial cells and goblet cells. Only a few areas of normal cultivated corneal epithelial cells were found. We suggest that the process of allo-graft rejection is responsible for the corneal epithelial destruction. We believe that this is followed by conjunctival invasion onto the amniotic membrane.

The Key issue is:The ultrastructure of removed cultivated epithelial transplants.