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S. Ahmad, S. Kolli, M. Lako, F. Figueiredo; The Differentiation of Human Embryonic Stem Cells Towards the Corneal Epithelial Lineage. Invest. Ophthalmol. Vis. Sci. 2007;48(13):478.
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© ARVO (1962-2015); The Authors (2016-present)
Human embryonic stem (hES) cells are pluripotent capable of differentiating into cells from all three germ layers. The adult stem cell (SC) microenvironment is important in maintaining their SC state. We investigated the differentiation of hES cells towards the corneal epithelial lineage by replicating factors within the corneal epithelial stem cell (CESC) microenvironment (i.e. basement membrane collagen IV and cytokines derived from limbal fibroblasts).
Limbal fibroblasts were cultured from cadaveric human limbal tissue (donated for research and obtained from UK Transplant). The expanded limbal fibroblasts were mitotically inactivated. Standard limbal epithelial medium was added to these mitotically inactivated limbal fibroblasts and conditioned for 24 hours. Tissue culture wells were plated with collagen IV. H1 [Wi-Cell] and hES-NCL1 [Newcastle University, United Kingdom] hES cells were then plated on the collagen IV using limbal fibroblast conditioned medium. The hES cells were cultured for 21 days. During this differentiation time period, the hES cells were analysed for the following: (1) morphological changes as assessed by phase contrast microscopy; (2) real time RT-PCR for the hES cell markers OCT4 and NANOG, the CESC marker p63, and the terminally differentiated corneal epithelial cell marker cytokeratin (CK) 3; and (3) flow cytometry for the hES cell marker SSEA4 and the CESC marker p63.
(1) A significant change in morphology of the hES cells was noted as early as the first 3 days of differentiation. The differentiated hES cells (from both hES cell lines) had significant areas with epithelial cell morphology. (2) Real time RT-PCR of the differentiated hES cells showed that both OCT4 and NANOG declined over the 21 days. The expression of p63 peaked at day 6, and CK3 peaked at day 15. (3) Flow cytometry showed that SSEA4 declined over the 21 days and p63 expression peaked at day 6. Both the real time RT-PCR and flow cytometry data showed significant differences between the two hES cell lines.
These experiments show that pluripotent hES cells can be differentiated towards the corneal epithelial lineage by replicating factors within the CESC microenvironment. Real time RT-PCR and flow cytometry show that the hES cell markers decline over the 21 day differentiation time period, p63 expression peaks at day 6, and this is followed by a peak in CK3 expression at day 15. Although both hES cell lines showed these trends, they did so to a varying degree.
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