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Alberto Muniz, Douglas Grow, Dallas Golden, Heuy-Ching Wang; Size of Embryoid Body Influences the Differentiation Efficiency of iPS into RPE. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3189.
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The efficiency of stem cell differentiation towards a desired lineage remains a challenge. Several studies have reported methods for differentiation of pluripotent stem cells into retinal cells. Here we investigate the differentiation efficiency of induced pluripotent stem cells (iPS) into retinal pigment epithelium (RPE) with different sizes of embryoid bodies (EBs) and spontaneous differentiation.
Human iPS (IMR-90 cells) were cultured and maintained with mTeSR1 on matrigel coated plates. The formation of 3-dimensional EBs with 100, 200 and 500 cell sizes (100 cells/EBs, 200 cells/EBs and 500 cells/ EBs) were prepared using AggreWell 400 plates for 24 hours and seeded on matrigel coated plates with differentiation media. Spontaneous differentiation was induced by differentiation media after 4 days of culture in mTeSR1. RPE cells were identified by morphology and pigmentation.
200 cells/EBs and 500 cells/EBs successfully attached to the coated plates, whereas 100 cells/EBs disintegrated in suspension. More cells with RPE morphology and pigmentation developed from 500 cells/EBs. Within 12 weeks of differentiation, the majority of the cells in the colonies of 500 cells/EBs developed monolayer sheets of hexagonal pigmented cells. The 200 cells/EBs adopted astrocyte morphology and never developed pigment. Small clusters of pigmented cells were derived from spontaneous differentiation by week 4. At 12 weeks some spontaneous differentiated clusters developed colonies of pigmented hexagonal cells without monolayer sheets. Pigmented cells were dissected, and continued in culture. The cells acquired a fibroblast-like appearance. After 4-6 weeks, cell density increased and the cells assumed a hexagonal shape and acquired pigmentation.
Our data demonstrates that the size of EBs can influence the differentiation efficiency of iPS into RPE. A platform to deliver uniform EB population by microwell technology can improve the differentiation efficiency of pluripotent stem cells into RPE needed for research and therapeutic applications.
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