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Xiaokun Wang, Julien Maruotti, Jose Roman, Haiquan Mao, Donald J Zack, Jennifer Elisseeff; The Effect of Collagen Vitrigel Nanostructure on Stem Cell Derived Retinal Pigment Epithelial Maturation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2360.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is the leading cause of blindness in the US in individuals over the age of 65 years. One uprising approach to obtain human RPE cells is to generate them from human pluripotent stem cells. These stem cell-derived RPEs could serve as cell resource for RPE transplantation. An optimal cell scaffold for tissue engineered RPE monolayer should simulate their natural microenvironment, support RPE maturation, along with favorable surgical properties, and biocompatibility. With the aim to develop an optimized tissue-engineered RPE monolayer, we investigated the influence of chemistry and morphology of collagen based materials to stem cell-derived RPE maturation in the current research.
Retinal pigment epithelial-like cells were differentiated from embryonic stem cells. In this study frozen differentiated cells were thawed and passed for 3 times before seeding to materials. For different collagen vitrigel (CV) groups, vitrification time varied from 3 days to 3 weeks at 40℃ in order to obtain different nanofibril structure. Beta-cyclodextrin collagen vitrigel (Beta CV) was prepared by mixing 5% β-CD solution with collagen solution then go through vitrification. PCL electrospun fibers were also prepared for RPE maturation evaluation. Cell morphology, gene expression were tested in all groups.
Longer vitrification time resulted in thicker collagen fibril (~100nm) and denser fibril density. CV3day has average fibril diameter of ~60-70nm, while CV3week has >100 nm fibril diameter. Beta-CV has no distinctive fibril structure and PCL electrospun fibers has diameter around 1 µm<br /> After 4-6 weeks culture, ES-RPE became pigmented and polarized, microvilli can be observed under SEM and TEM among all groups. qPCR results showed significant enhancement of ES-RPE maturation on CV-3D group, which indicates the nanofibril scale of CV3day group is essential for RPE maturation
In this study, we evaluated the ES-derived RPE maturation on different substrates, including synthetic polymer and collagen-based materials. RPE proliferate well on different substrates and became mostly pigmented and polarized between 4~6 weeks. Quantitative study by qPCR showed RPE characteristic marker expression significantly increased in CV3day group. This study provided an insight of how nanofibril in collagen based material influence RPE maturation.
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