Purchase this article with an account.
Anni E. Sorkio, Tanja H. Ilmarinen, Joachim S. Loo, Heli T. Skottman; Microparticles in Differentiation of Retinal Pigment Epithelial Cells from Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5914.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) has a crucial role in the support and function of the retina and its photoreceptors. Human pluripotent stem cells (hPSCs) have been shown to differentiate into RPE cells, but the current differentiation protocols are inefficient and result in low yields. The current culture methods of hPSCs on feeder cells or in two-dimensional feeder-free systems are laborious and prevent the production of stem cells in large-scale. The aim of this study, was to investigate if biodegradable poly(L-lactide acid) (PLLA) and commercial cellulose anion exchange diethylaminoethyl (DE) 53 microparticles could be exploited in increasing the efficacy of differentiation of hPSCs into RPE cells. In addition, the potential of the microparticle culture system for hPSCs maintenance in three-dimensional feeder-free conditions was evaluated.
PLLA microparticles were prepared through a single emulsion method. In order to increase the cell attachment on microparticles, the particles were coated with extracellular matrix proteins. The hPSC were adapted in two-dimensional feeder-free cultures before seeding cells on microparticles. The attachment, proliferation, and maintenance of pluripotency of hPSCs on self-prepared and commercial particles were investigated by immunofluorescence stainings. Spontaneous differentiation of hPSCs was induced by removing basic fibroblast growth factor from the culture medium. The appearance of pigmentation and expression of RPE maturation markers on gene expression levels were examined.
The self-prepared PLLA microparticles did not support the attachment and proliferation of stem cells. In contrast, hPSCs attached, proliferated and maintained their pluripotency on DE 53 particles in feeder-free culture conditions. The cell clusters on DE 53 microparticles acquired pigmentation after 15 days of differentiation. In addition, the cells expressed RPE maturation markers after 35 days of differentiation.
Our data indicates that the commercial DE 53 microparticles support the maintenance of stem cells in feeder-free culture conditions. Furthermore, hPSCs can be successfully differentiated into RPE cells in microparticle suspension cultures.
This PDF is available to Subscribers Only