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Xinbo Li, Yufeng Wang, Davey Li, Ted S Acott, Mary J Kelley; Connexin43 is a key element for differentiation of iPSCs to trabecular meshwork like cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3530.
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© ARVO (1962-2015); The Authors (2016-present)
Primary open angle glaucoma (POAG), a blinding optic neuropathy, often results from elevated intraocular pressure due to obstruction of aqueous humor outflow via the trabecular meshwork (TM). Reduced TM cellularity has been reported in POAG. Our previous work and that of others also showed that induced pluripotent stem cells (iPSCs) can differentiate to a TM-like cell, but the underlying mechanisms are not fully elucidated. Gap junctions play pivotal roles in maintaining cell differentiation and cell proliferation. Therefore, we investigated the gap junction protein connexin43 (Cx43) expression in iPSCs, embroyoid bodies (EBs), TM and scleral cells, as well as the effect of shRNA knockdown of Cx43 on the differentiation of iPSCs to TM like cells.
TM and iPSC cell culture, TM and scleral tissue sections, and perfused anterior segment organ culture with microspheres were used, in addition to Western immunoblotting (WB), immunofluorescence (IF), lentiviral transduction, and shRNA silencing of Cx43 after EBs production.
IF showed strong punctate labeling of Cx43 in cultured TM cells, iPSCs, and TM tissue sections when compared to cultured scleral cells and tissue sections. Cx43 protein expression was significantly increased in lysate from both cultured TM cells and tissues compared to cultured scleral cells as shown by WB (P<0.01). Additionally, Cx43 was highly expressed in iPSCs and EB cells. Using an EGFP lentiviral vector, we found that lentiviral transduction can reach nearly 100% efficiency in HEK293 and HTM cells. Knockdown of Cx43 expression in HTM cells using lentivirus-delivered Cx43 shRNA led to an 80%-90% reduction of Cx43. The differentiation of iPSCs to TM-like iPSCs was significantly blocked by administration of lentiviral Cx43 shRNA, after EBs production. This blockage was demonstrated by the reduced expression of TM markers CHI3L1 and MGP noted in lentiviral Cx43 shRNA treated groups (P<0.01) by WB and IF in three independent experiments.
Cx43 is highly expressed in TM cells and tissues. Blockage of iPSC differentiation to TM-like iPSCs suggests that Cx43 may play a key role in the differentiation. Understanding the mechanism of differentiation of stem cells to a TM-like cell may aid in furthering future regenerative cell therapies to treat POAG.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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