Quantitative pigment extraction analysis for human pluripotent stem cell derived retinal pigment epithelial cells

摘要

Pigmentation is a typical feature for retinal pigment epithelial cells (RPE), and each RPE cell represents individual characteristics. Although, type and intensity of pigmentation may vary, still most cells are pigmented to some extent. In this study, we set-up a standardized quantitative melanin content analysis for human embryonic stem cell (hESC) derived RPEs (hESC-RPE) and compared this method to an image based pigment quantification technique. The optical quantification of pigmentation was done from micrographs of hESC-RPE. The extracted pigment was quantitated with spectral analysis, with UV-Vis spectroscopy, and the size-distribution with dynamic light scattering. The results revealed that the measured particle sizes of extracted melanin corresponded to known sizes extracted mela-nosomes. In addition, the optical pigmentation and absolute melanin concentration were clearly correlating. Our data suggests that image analysis and quantitative melanin content analysis can be interchangeably utilized in pigmentation quantitation: if the relative pigmentation of hESC-RPE cells is desired to be estimated with a non-invasive method then image analysis is the choice of method, but if the exact amount of melanin needs to be evaluated, then the new melanin extraction method presented here, should be chosen.