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S. Balasubramanian, I. Ahmad; Examination of Induced Pluripotency of Limbal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6066.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelium is renewed by stem cells located in the basal layer of the circular limbal epithelium between cornea and conjunctiva. We have demonstrated that limbal stem cells when removed from their niche and cultured in the presence of growth factors (EGF and FGF2) display neural stem cell properties and potential (Zhao et al., 2002, Dev. Biol. 250:317). Given the morphological similarities of neurospheres generated by limbal stem cells in the presence of growth factors to ES cell colonies and the plasticity demonstrated by these cells we were interested in knowing if these cells acquire pluripotent properties in vitro.
Cell dissociates from limbal epithelium were cultured in the presence of EGF and FGF2 for the generation of clonal neurospheres. The cells in neurospheres were examined for the expression of putative limbal stem cell markers and the activation of core genes defining pluripotency. Cells in the neurospheres were examined for their differentiation along all three germ cell lineages. Here, we examined, in detail their ability to generate neurons with functional properties and differentiate along different cell-type specific lineages as a test of the fidelity of their plastic nature.
The examination of neurospheres revealed that they consisted of proliferating cells expressing both neural (Nestin) as well limbal (α-enolase and p63) stem cell/progenitor-specific genes. Next, we analyzed different batches of neurospheres cultured in EGF+FGF2 and EGF+FGF2+Noggin for the expression of transcripts corresponding to transcription factor genes for pluripotency i.e., Oct4, Klf4, Sox2, c-myc (Takahashi and Yamanaka, 2006, Cell 126:663). We also examined the expression of Nanog. In all batches of neurospheres we detected the expression of Sox2, Klf4 and c-myc. In one batch of neurospheres expression of Oct4 and Nanog was also detected, however, their expression levels were remarkably low in comparison to that of Sox2 and Klf4. That the plasticity of the limbal stem cells was directly influenced by the microenvironment was further demonstrated by their display of functional neuronal properties only in the presence of neonatal neuronal progenitors.
Our preliminary results suggest that the limbal cells, exposed to growth factors, acquire plasticity greater than previously demonstrated, i.e., they may be induced by culture conditions into pluripotent ES cell-like cells.
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