The Mechanism of Ex Vivo Expansion of Limbal Stem Cells and Clinical Applications


Ray Jui-Fang Tsai *,

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The successful clinical application of ex vivo expansion of limbal Stem Cells has been documented for more than 10 years. The limbal stem cells harvested in this way are effective for ocular surface reconstruction, especially in autologous transplantation. Briefly, limbal stem cells were taken from normal fellow eye of the patient and cultured in vitro for two weeks, then transplanted to the diseased eye to restore patient’s vision. Therefore, to understand the mechanism of ex vivo expansion of limbal stem cells is important to further investigation of ex vivo expansion of other adult stem cells and their future clinical applications. In order to understand the mechanism of how the human limbal stem cells can repopulate over the limbal deficient corneal surface and maintain its clarity for over 10 years, we studied the label retention cells (LRC) of limbal epithelium with BrdU staining and confirmed the existence of stem cells with P63 staining in our culture system. The explanted culture of limbal tissue was placed on the cryo-preserved amniotic membrane in which the amniotic cells were still preserved. Culture medium with 5% patient’s serum was changed every other day. 3T3 was not used as feeder layer or co-culture. PCNA staining confirmed that limbal epithelial cells proliferated better on amniotic membrane than on the plastic plate. For label retention cells (LRCs) study, BrdU was fed to limbal explanted culture continuously for one week, and then followed by chasing the BrdU and transplanted the cultured limbal stem cells with amniotic membrane into nude mice for another week. Basal layer of cultured limbal epithelium on amniotic membrane was stained positively by anti-BrdU antibody. P63 staining was also positive. These positive results confirmed that the basal cells of limbal epithelial cells on amniotic membrane were stem cells. In this system, amniotic membrane worked as niches for limbal stem cells proliferation and differentiation. I also reviewed 54 cases that the allograft limbal stem cells had been performed during 1998 to 2000. Nineteen cases had been followed for more than 18 months were studied. There were 6 cases of chemical burn, 7 cases of OCP, 3 cases of SJ syndrome, and 3 cases with chronic inflammation. The success rate was 9/46(47.4%), including chemical burn 4/6(66.7%), OCP 3/7(42.9%), SJ syndrome 0/3 (0%), and inflammation 2/3 (66.7%). The vision improvement was 65%. From 2001, I changed the immunosuppression regimen: prednisone 0.5mg/kg/d, cyclosporine A 2.5 mg/kg/d BID, cellcept 2g/d BID. 15 patients were included for studied. The success rate improved to 11/15(73.3%), including Chemical burn: 4/7(57.1%), OCP: 4/5(80%), SJ Syndrome 2/2(100%), and inflammation 1/1(100%). In conclusion, in our culture system, limbal explanted culture on amniotic membrane provides a practical method for ex vivo expansion of limbal stem cells. The stem cells harvested can be transplanted to human diseased cornea and they can reserve their stem cell function for more than 10 years in autograft, but the survival rate for allograft was 50% to 70 % which depends on the immunosuppression regimen were used.