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Q. Sun, J. Hua, C. Malcuit, L. Vilner, L. Lemieux, I. Klimanskaya, I. K. Sugino, M. A. Zarbin; Fetal RPE (fRPE) vs. Human Embryonic Stem Cell-Derived RPE (hES-RPE): Integrin mRNA Expression and Behavior on Aged Bruch’s Membrane (BM). Invest. Ophthalmol. Vis. Sci. 2008;49(13):3564.
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© ARVO (1962-2015); The Authors (2016-present)
To determine: 1) whether the degree of maturation can affect hES-RPE mRNA integrin expression compared to that of fRPE and 2) whether integrin mRNA expression can predict behavior on BM.
3 batches of hES-RPE (hES passage 51 + 2 passages following RPE differentiation) of different degrees of pigmentation were generated by varying culturing conditions [culture time (6- 10 wks.; media ± serum & growth factors]. hES-RPE or fRPE (2 lines, P1-4, 4-9 days in culture) were seeded onto the inner collagenous layer (ICL) of peripheral BM explants (9 donor pairs; age 75.7±9.89 yrs.). Explants were harvested following 1 or 21 days in culture and surface morphology analyzed with scanning electron microscopy (SEM). Remaining cells from BM seeding were analyzed with real time PCR for integrins (α(1-6, v), β(1,3 4-6)) and RPE markers, Bestrophin and MITF.
In all cell preparations, the most abundant α integrin was αv; the most abundant β integrin was β1. All cells showed very little β6. The 2nd most abundant integrin in the least pigmented hES-RPE was α3. Similarly, 6/7 fRPE preparations showed high levels of mRNA for α3. The mid and high level pigmentation batches showed similar integrin profiles with α6 as the 2nd most abundant. This was similar to 1/7 fRPE. Bestrophin and MITF mRNA levels increased with hES-RPE of increasing pigmentation. Bestrophin and MITF levels were highly variable in fRPE. At day-1, SEM showed poor attachment and spreading of all hES-RPE preparations compared to fRPE. At day-21, SEM showed significantly increased resurfacing by hES-RPE compared to day-1 with the degree of resurfacing and cell surface morphology similar to that of fRPE.
Despite high levels of integrins important for attachment to BM, hES-RPE do not attach as rapidly to the ICL as fRPE and, regardless of the low attachment seen at day-1, hES-RPE showed similar resurfacing to fRPE at day-21. These results indicate that additional factors may be important in determining cell behavior on BM.
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