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Hossein Nazari Khanamiri, Danhong Zhu, Christine Spee, Mark Humayun, David Hinton; Transforming growth factor beta secretion by human embryonic stem cell derived retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2208. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor beta (TGF-β), produced by retinal pigment epithelium (RPE), is a major component of the subretinal immune suppressive microenvironment. Implantation of human embryonic stem cell derived RPE (hESC-RPE) is emerging as a new strategy for the replacement of lost RPE cells in age-related macular degeneration. It is important to know whether these implanted hESC-RPE cells can produce cytokines involved in subretinal immune privilege environment. In the current study, we analyzed the ability of polarized hESC-RPE to produce TGF-β1 and -β2.
The H9 human embryonic stem cell line was used for derivation of RPE cells. hESC-RPE polarization (trans epithelial resistance above 300 Ohm/cm2) was achieved by long-term growth on permeable inserts. Apical and basal production of active TGF-β1 and -β2 was compared by ELISA in the medium collected from the apical and basal compartments. Total TGF-β1 and -β2 were measured by ELISA after acid activation. The absolute amount of secreted TGF-β was normalized to cellular total protein. Intracellular localization of the TGF-β1 and -β2 was visualized by immunostaining and confocal microscopy.
The TGF-β1 was hardly detectable in the medium of polarized hESC-RPE. The level of latent (inactive) TGF-β2 was higher in the medium of the basal side, compared to the apical side of the hESC-RPE cells. However, the level of active TGF-β2 was higher in the medium from the apical side than the medium from the basal side. Active TGF-β2 was the major form of TGF-β2 secreted from the apical side, reaching to about 50% of total apically secreted TGF-β2.
TGF-β2 is the main TGF isoform secreted by hESC-RPE. More active TGF-β2 is secreted from the apical side of the polarized hESC-RPE, while more latent TGF-β2 is secreted from the basal side. Apically secreted active TGF-β2 may play a role in providing an immunosuppressive microenvironment for the implanted hESC-RPE cells.
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