Human limbal progenitor cell characteristics are maintained in tissue culture.
- Author:
Shaohui LIU
1
;
Jing LI
;
Chuanfu WANG
;
Donald TAN
;
Roger BEUERMAN
Author Information
- Publication Type:Journal Article
- MeSH: Cell Differentiation; Coculture Techniques; Connexin 43; metabolism; Cornea; cytology; Epithelial Cells; Humans; Immunohistochemistry; Keratins; metabolism; Limbus Corneae; cytology; Protein Precursors; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; cytology; Tissue Culture Techniques
- From:Annals of the Academy of Medicine, Singapore 2006;35(2):80-86
- CountrySingapore
- Language:English
-
Abstract:
INTRODUCTIONTo determine the differentiation of human limbal epithelial cells in tissue culture.
MATERIALS AND METHODSEpithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR).
RESULTSThe colony-forming efficiency of primary cultured (P0) cells was about 19.35 +/- 6.46% (mean +/- SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P <0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 +/- 1.27)% (n = 7, P0) and (3.96 +/- 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse.
CONCLUSIONSHuman limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.