The Effect of Human Embryonic Stem Cell Seeding Density on the Timing and Yield of Differentiation into Retinal Pigment Epithelium

Abstract

Purpose: : Functional Retinal Pigment Epithelium (RPE) can be generated from Human embryonic stem cells (hESC). These cells hold great promise as a potentially unlimited source of material for the treatment of retinal disorders such as age related macular degeneration. In this study we have investigated hESC seeding density as a source of variability in the differentiation process. HESC require manual passaging and are very sensitive to the timing and method of passage; this leads to considerable variation in cell seeding density which is difficult to measure and control. Here we have employed a novel cell imaging technique to quantify both the cell seeding density and the yield of pigmented RPE.

Methods: : Cells from the hESC line Shef 6 were mechanically passaged at various densities onto mitotically inactivated mouse embryonic feeder cells. The percentage confluency at the beginning of the differentiation was measured by imaging the whole flask on a modified scanning device. After a 50 day differentiation protocol, the RPE yield was assessed by measuring pigmentation as a percentage of the surface area and by quantitative PCR.

Results: : Shef6 hESC were passaged at different split ratios; at day 7 the seeding density (% confluency) varied between 10% and 48% (n=9). Flasks seeded with a high density of hESC reached a maximum pigmentation of 3.6% at day 50 where as a low hESC seeding density produced a maximum of 0.07% (as determined by image analysis). This corresponded to 2 and 104 individual pigmented-RPE foci respectively that were deemed large enough to harvest manually. There was a strong linear correlation between hESC seeding density and the natural logarithm of percentage pigmentation at day 50 (R² = 0.848).

Conclusions: : Seeding density is rarely controlled for in hESC differentiation experiments due to the inherent variability brought about by mechanical passaging. Here we show that this often neglected parameter has a significant impact on the timing and yield of differentiation of hESC into RPE cells, with a strong positive correlation between seeding density and RPE yield. The mechanism by which seeding density influences RPE differentiation is unclear but this correlation may be important in the design of an efficient and economical hESC-RPE manufacturing process.

Keywords: retinal development • retinal culture • age-related macular degeneration