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Clara Iglesias, Akshayalakshmi Sridhar, Sarah Ohlemacher, Jason S Meyer; EXAMINATION OF GAP JUNCTION INTERCELLULAR COMMUNICATION IN HUMAN PLURIPOTENT STEM CELL-DERIVED RETINAL PIGMENT EPITHELIUM. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3998.
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Retinal pigment epithelial (RPE) cells serve to support retinal photoreceptors and are adversely affected in many blinding disorders such as AMD. While many current strategies are focused on the replacement of RPE cells using hPSCs, the demonstration of the proper functionality of these stem cell-derived RPE remains incomplete. Previous studies have identified gap junctions as important for communication between RPE cells in vivo. However, there are limited studies testing the functionality of these cells derived from a hPSC source. Thus, efforts were undertaken to examine the role of gap junction proteins in intercellular communication in hPSC-derived RPE.
To initiate differentiation hPSCs were grown in a medium consisting of DMEM/F12 (1:1) containing 20% knockout serum replacement, L-glutamine, and MEM non-essential amino acids. Within eight weeks of differentiation, RPE cells were readily identified by their hexagonal cobblestone shape and accumulated pigmentation. RPE was isolated by microdissection and expanded in the presence of FGF-2, EGF and heparin. To confirm the identity of these cells, the expression of genes and proteins characteristic to RPE were analyzed using both RT-PCR and immunocytochemistry.
hPSC-derived RPE cells expressed characteristic markers, such as MITF, RPE65 and ZO-1, as assessed by RT-PCR and immunocytochemistry. The expression of gap junction genes within these hPSC-derived RPE cells was then examined by RT-PCR, in which the most prevalent gap junction gene was found to be GJA1, encoding for the protein Cx43. Subsequent immunocytochemistry analysis confirmed the presence of Cx43 at points of contact between neighboring RPE cells. Current studies are underway to test the ability of hPSC-derived RPE to effectively communicate through gap junction channels.
hPSC-derived RPE are demonstrated to possess a large complement of native RPE-associated characteristics, including the expression of gap junction proteins. The presence of these proteins will likely be important for the maturation and functionality of these RPE cells. The results of these studies serve to provide a greater understanding of hPSC-derived RPE functionality, which may be essential for the development of translational applications for these cells.
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