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Jin Yang, Yi Lu; The dosage effect of pax6 in lens epithelial cells induction from iPSCs. Invest. Ophthalmol. Vis. Sci. 2013;54(15):464.
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Lens development is a complex process in which a single epithelial layer undergoes several stages of competence, induction and differentiation, ultimately forming a highly specialized organ. Our previously study identified a highly efficient procedure for generating lens-like cells from cataract patient lens epithelial cells (HLECs)-derived iPSCs, however, the percentage of induced lens epithelial cells is low(figure 1). The transcription factors pax6 has been shown to be essential for lens induction, but it’s roles during LECs differentiation remain unknown. So we try to detect the function of Pax6 in the lineage tracing of lens specific differention.
First we construct a new Sox2-RFP/LEP503-GFP dual fluorescence reporter system. Then we performed a 3-step lens induction procedure on HLECs-derived iPSCs. Finally we reduce Pax6 expression with shRNA at different induction stage , assay the percentage of GFP-positive cells and the relative lens gene expression(six3,CRYAA,CRYAB etc.).
Sox2 promoter was active in iPSCs, and LEP503 promoter was specifically actived in HLECs based on our previously study, and HLECs-iPSCs transduced with lenti-sox2-RFP/LEP503-GFP express RFP, we found RFP was reduced gradually and GFP emerge and was increased( figure2)in this process. For study the role of Pax6 during HLECs induction. Pax6 was inhibited with shRNA, and we found Pax6 is most needed in stage 2, the 50% reduction of pax6 in stage 2 will lead to 90% HLEC diminish, and overexpress of Pax6 at this stage will enhance HLEC differentiation.
We establish a lenti-sox2-RFP/LEP503-GFP system, which can dynamically trace HLEC differentiation relative to the dosage of Pax6 in 3 stage induction ,and we found stage 2 is the key stage for Pax6 function, overexpress Pax6 will enhance HLEC induction.
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