One for all: A standardized protocol for ex vivo culture of limbal, conjunctival and oral mucosal epithelial cells into corneal lineage

Background aims Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. Methods Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers ( ABCG2 and P63α ) and differentiation markers ( CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43 ). Fluorescence-activated cell sorter analysis was performed for ABCG2. Results The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3 , CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. Conclusions Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.