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Kati M Juuti-Uusitalo, Jussi Muranen, Helena Lähdekorpi, Elina Pajula, Hannu M T Uusitalo, Kai Kaarniranta, Heli Skottman, ; An in vitro model of outer blood-retinal barrier based on human embryonic stem cell derived retinal pigment epithelial cells and human retinal microvascular endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2339.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this study was to create an in vitro outer blood-retinal barrier (oBRB) cell co-culture model using highly pigmented and mature human embryonic stem cell (hESC) -RPEs and human retinal microvascular endothelial cells (hREC). The long-term co-culture effects on the barrier properties, morphology and function of hESC-RPE cells was further assessed.
The hESC- RPE cells (Regea 08/017, Regea 08/023 or Regea 11/013 cell lines) were seeded on other side of perforated polyester filter inserts than primary human retinal microvascular endothelial cells (ACBRI 181). These were co-cultured together up to 6 weeks. The barrier function was examined by measuring the trans-epithelial resistance (TER) using a voltohmmeter and the permeability of a 4 kDa fluorescein isothiocyanate-dextran (FD4) using the Ussing chamber system. The expression and the localisation of RPE and endothelial marker proteins were analysed with the confocal microscopy, and the fine structure with transmission electron microscopy (TEM). The effects of the co-culture on growth factor secretion of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were analysed with ELISA.
The TER assessment revealed that co-culture increased the barrier function in hESC-RPE cells. The co-culture had only a modest effect on the cumulative permeability of FD4 in hESC-RPE. In confocal microscopy we saw that RPE specific cellular retinaldehyde-binding protein and endothelial cell specific von Willebrand factor had cell type specific localisation. TEM revealed a cell line specific effect on the extracellular matrix deposition. The effect of co-culturing on VEGF and PEDF secretion was also cell line specific. VEGF secretion in RPE cells differentiated from Regea 08/023 cell line but increased in Regea 08/017 and Regea 11/013 cell lines. The secretion of PEDF was increased after co-culture in RPE cells differentiated from Regea 08/017 and Regea 08/023 but not in Regea 11/013 cell line.
The long-term co-culture of hESC-RPE cells with hRECs has effects on the matrix deposition, barrier function and the functionality of hESC-RPE cells. Furthermore the data suggests - hESC-RPE line specific differences in growth factor secretion.
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