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Yingting Zhu, Wen-Juan Lu, Shuangling Chen, Sean Tighe, Yuan Zhang, Chen-Wei Su, Hua He, Scheffer C G Tseng; Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5277. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To further explore the mechanism of reprogramming human corneal endothelial (HCEC) monolayers into neural crest (NC)-like progenitors by knockdown with p120-catenin (p120)-Kaiso siRNAs.
HCEC monolayers derived from collagenase digestion of stripped Descemet membrane were cultured to 7 days and treated with 100 nM of each p120 and Kaiso siRNAs with or without various inhibitors or siRNAs in MESCM for up to 5 weeks. Before termination, cells were further treated with 10 μM BrdU for 4 h. Transcript expression of NC, cell cycle and senescence markers, JAKs and STAT3 was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of BrdU, NC, cell cycle and signaling markers. Western blotting was used to measure relative protein level.
We have reported contact-inhibition of HCECs can be delayed from Day 21 to Day 42 by switching the culture medium from SHEM to MESCM, a serum-free and LIF/bFGF-containing medium, by transient activation of LIF-JAK1-STAT3 signaling with concomitant inhibition of eventual nuclear translocation of p16INK4a. Using the latter system, we have created a novel tissue engineering technique by implementing 5 weekly knockdowns with p120-Kaiso siRNAs to achieve effective expansion of HCEC monolayers to a transplantable size with a normal density by reprogramming HCECs into neural crest progenitors through activating p120-Kaiso-RhoA-ROCK-canonical BMP signaling. Interestingly, a single knockdown with p120-Kaiso siRNAs at Day 42 failed to achieve such reprogramming when contact inhibition transitioned to senescence with nuclear translocation of p16INK4a. In contrast, 5 weekly knockdowns with p120 and Kaiso siRNAs precluded senescence mediated by p16INK4a by induction of nuclear translocation of Bmi1 that downregulated p16INK4a and prohibited nuclear translocation of p16INK4a. Such nuclear translocation of Bmi1 was resulted from sustained activation of JAK2-STAT3 signaling downstream of p120-Kaiso-RhoA-ROCK signaling. In addition, STAT3 or Bmi1 siRNA impeded nuclear exclusion of p16INK4a and suppressed BrdU labeling and nuclear translocation of Oct4, Sox2 and Nanog induced by p120-Kaiso siRNAs.
We have discovered another important mechanism of the reported engineering strategy of HCECs that lies in prevention of senescence mediated by nuclear translocation of p16INK4a.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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