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Yingting Zhu, Xin Liu, Wenjuan Lu, Szu-Yu Chen, Scheffer C G Tseng; Activation of RhoA-ROCK-BMP-miR302 Network Reprograms Human Corneal Endothelial Cells to Neural Crest Progenitors . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1143.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the mechanism of reprogramming human corneal endothelial cell (HCEC) monolayers into neural crest (NC) progenitors by knockdown with p120-catenin (p120)-Kaiso siRNAs.
HCEC monolayers derived from stripped Descemet membrane were cultured to 7 days and treated with 100 nM of p120 and Kaiso siRNA in MESCM for up to 5 weeks. Before termination, cells were labeled with 10 μM BrdU for 4 hours. Transcript expression and cytolocalization of NC, embryonic stem cell (ESC), keratocyte, and cell cycle markers, miR 302s, and signaling markers was determined by RT-qPCR and immunostaining. Western blotting was used to measure respective proteins.
As reported previously, MESCM promoted expansion the size of HCEC monolayers to 4.4 ± 0.6 mm without but to 11.0 ± 0.6 mm in diameter with weekly knockdown by p120-Kaiso siRNAs from 1/8 corneoscleral rim after 6 weeks of culture. The use of MESCM medium containing LIF was important because LIF promoted expansion of HCEC monolayers by delaying contact inhibition via activation of LIF-JAK-STAT3 signaling and through downregulating senescence-related p16 while upregulating cell cycle-promoting genes. Addition of p120-Kaiso siRNA after 1-2 weeks of culture could further promote expansion by reprogramming HCECs into their progenitors through activation of RhoA-ROCK-canonical BMP signaling. Further analysis indicated that activation of canonical BMP was required for reprogramming because knockdown of BMP receptors abolished such reprogramming, but alone was not sufficient to lead to such reprogramming since addition of BMP4/6 (25 and 50 ng/ml, respectively) activated canonical BMP signaling, promoted expression of ESC and NC markers but did not lead to nuclear translocation of Oct4-Sox2-Nanog complex. Canonical BMP signaling required RhoA-ROCK signaling triggered by knockdown with p120-Kaiso siRNAs to activate miR 302b/c-Oct4-Sox2-Nanog network for reprogramming. Finally, the NC progenitor status was confirmed by differentiation of HCEC progenitors into keratocan-expressing keratocytes.
This new strategy of reprogramming of HCECs into NC progenitors by activation of RhoA-ROCK-BMP-miR 302 Network could be used to engineer HCEC surgical grafts containing HCECs for endothelial keratoplasties to meet a global corneal shortage in treating corneal blindness caused by corneal endothelial dysfunction.
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