Ex vivo expansion and transplantation of limbal epithelial stem cells.
OBJECTIVE
To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells.


METHODS
Prospective, noncomparative, interventional case series.


METHODS
Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye).


METHODS
Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy.


METHODS
Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy.


RESULTS
The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001).


CONCLUSIONS
These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.
Shortt
Alex J
AJ
Cells for Sight Transplantation and Research Programme, UCL Institute of Ophthalmology, London, United Kingdom.
Secker
Genevieve A
GA
Rajan
Madhavan S
MS
Meligonis
George
G
Dart
John K
JK
Tuft
Stephen J
SJ
Daniels
Julie T
JT
D000328:Adult; D000368:Aged; D002452:Cell Count; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D019573:Epithelium, Corneal / Q000166:cytology*; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D011446:Prospective Studies; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D014182:Transplantation, Autologous; D014184:Transplantation, Homologous; D016896:Treatment Outcome; D014792:Visual Acuity
D007166:Immunosuppressive Agents