Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction

Abstract

PurposeTo investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET) using human amniotic membrane for corneal limbal stem-cell deficiency.MethodsProspective, noncomparative case series. Eighteen patients (19 eyes) with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed.ResultsCorneal epithelium cultivated on amniotic membrane (two to four layers) was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7%) of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47). A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2%) eyes. Clinical failures occurred in five (26.3%) of 19 eyes, and histopathologic failures occurred in seven (36.8%) of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7%) eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31) of follow-up. Complications included infectious keratitis (three cases) and recurrent symblepharon (one case). All complicated cases had lid abnormalities. Factors affecting the final clinical outcomes were lid abnormalities, abnormal corneal stromal beds, and complications.ConclusionCLET can successfully restore ocular surface damage in most cases with corneal limbal stem cell deficiency.

Limbal Stem Cells from Aged Donors Are a Suitable Source for Clinical Application

“…Our results fit with these findings, as LSC from donors older than 60 years showed higher levels of the putative stem cell marker p63. As assayed by WB, immunofluorescence, and qPCR, the LSC from each donor group were characterized, according to previous well-stablished statements [30], as they do not express corneal differentiation markers (CK12, CK3, Cx43, and E-cad) and express CK19, that despite being considered a conjunctival marker it is also expressed by LSC [28, 29, 43, 44]. Nevertheless, LSC from donors younger than 60 years expressed p63 cells at a percentage <3% as tested by Q-IF, consistent with the results of qPCR and WB that also showed lower levels of this putative stem cell marker.…”

Human corneal cell culture models for drug toxicity studies

“…In the human eye, cell-based and scaffold-based corneal engineering have been used in corneal transplantation [78]. For example, grafts of autologous limbal stem cells on fibrin [79] or on amniotic membrane [80, 81] have been used to restore the damaged corneal surface. In animal models, collagen, amniotic membrane with gelatin, and poly( N -isopropylacrylamide) (PNIPAAm) have been used for tissue engineering of corneal endothelium [82].…”

Clinical Outcomes From Cultivated Allogenic Stem Cells vs. Oral Mucosa Epithelial Transplants in Total Bilateral Stem Cells Deficiency

“…The tissue from a cadaveric donor was obtained within 24 h (22) to 7 days post-mortem (28). One study obtained cadaveric tissue within 2-3 weeks before the planned allogenic CLET procedure (24). Basu Most culture protocols used 3T3 feeder cells in order for the graft to receive proper nutrients, but they also have a role in detoxifying the culture media and providing extracellular matrix proteins (44).…”

“…The cultivated limbal stem cells were transferred onto the ocular surface on amniotic membrane, which was secured with sutures in all studies with two exceptions, Zakaria et al (27), who secured the graft with fibrin glue, and Ganger et al (29), who used both, either sutures or fibrin glue. In some studies a bandage contact lens was inserted after the transplant (21,24,(26)(27)(28)(29)31) while in other studies a second amniotic membrane was used as a patch (18,22,27,34). One study mentioned using either bandage contact lens or amniotic membrane (20) whereas one study does not mention the use of either (17) In cases of symblepharon, fibrovascular tissue surrounding rectus muscles was dissected in order to achieve normal ocular movement (31).…”