Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.
OBJECTIVE
The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer.


METHODS
Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12).


RESULTS
The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions.


CONCLUSIONS
The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.
Balasubramanian
Sudha
S
L & T Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
Jasty
Srilatha
S
Sitalakshmi
Guruswamy
G
Madhavan
H N
HN
Krishnakumar
Subramanian
S
D016475:3T3 Cells; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D018031:Connexin 43 / Q000032:analysis / Q000235:genetics; D019573:Epithelium, Corneal / Q000737:chemistry / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000737:chemistry*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C081876:CKAP4 protein, human; D018031:Connexin 43; D008565:Membrane Proteins; D009363:Neoplasm Proteins