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Citation: Kamao H, Mandai M, Wakamiya S, et al. Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE. Invest Ophthalmol Vis Sci. 2014;55:8309-8318. DOI:10.1167/ iovs.14-14694 PURPOSE. For the transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), determination of the maturation status of these cells is essential, and the degree of pigmentation (dPG) can serve as a good indicator of this status. The aim of this study was to establish a method of objectively and quantitatively evaluating the dPG of hiPSC-RPE. METHODS.Two observers determined the dPG subjectively by observing recorded images of hiPSC-RPE as follows: the dPG of a single cell was classified into three different pigmentation stages, and the overall dPG was compared between two cell groups to identify the group with the higher dPG. The j statistic was applied to assess interobserver reproducibility. Next, the dPG of single cells and cell groups was objectively determined by the lightness of the hue, saturation, and value (HSL) color space, and the correlation between the subjective evaluation and time-dependent change in the objective dPG of hiPSC-RPE was investigated.RESULTS. The j statistic was 0.88 and 0.81 in the single-cell and cell-group observations, respectively. The objective dPG of single cells and cell groups was highly correlated with the subjective dPG. However, the observers were occasionally unable to subjectively determine the group with the higher dPG. The objective dPG increased in a time-dependent manner.CONCLUSIONS. The lightness of the HSL color space can be used to objectively and quantitatively evaluate the dPG of hiPSC-RPE in culture. The objective evaluation was consistent and was able to better identify small differences than subjective evaluation.Keywords: retinal pigment epithelium, degree of pigmentation, human induced pluripotent stem cell A ge-related macular degeneration 1 (AMD) is a leading cause of severe visual loss in developed countries, and the agerelated degeneration of the retinal pigment epithelium (RPE) is considered an essential contributing factor to the disease pathogenesis. The RPE, a monolayer of pigmented cells, lies between the neural retina and the choroid and plays essential roles in the maintenance of photoreceptors 2 and choroidal vessels. Antivascular endothelial growth factor (VEGF) therapies have significant effects on wet AMD patients; however, recurrent cases, nonresponders, and dry AMD patients do not gain much benefit from current therapies. Retinal pigment epithelium transplantation for AMD for the purpose of replacing degenerated RPE with healthy RPE was introduced more than 2 decades ago and has attracted attention as an alternative therapy in recent years. To date, transplantation of allogeneic fetal RPE 3 or autologous peripheral RPE 4 has been reported as clinical therapy for AMD patients, although neither represents an ideal tissue source; the former has caused immune rejection, while t...

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