April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Author Affiliations & Notes
  • Clara Iglesias
    Biology, IUPUI, Indianapolis, IN
  • Akshayalakshmi Sridhar
    Biology, IUPUI, Indianapolis, IN
  • Sarah Ohlemacher
    Biology, IUPUI, Indianapolis, IN
  • Jason S Meyer
    Biology, IUPUI, Indianapolis, IN
    Stark Neuroscience Research Institution, Indiana University, Indianapolis, IN
  • Footnotes
    Commercial Relationships Clara Iglesias, None; Akshayalakshmi Sridhar, None; Sarah Ohlemacher, None; Jason Meyer, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3998. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 721 stem cells • 701 retinal pigment epithelium • 500 differentiation  

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