Purchase this article with an account.
Hiroyuki Kamao, Michiko Mandai, Katsutoshi Goto, Junichi Kiryu, Masayo Takahashi; Evaluation of Degree of Pigmentation as an Indicator of Maturation status in Human iPSC-RPE. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1364.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In transplantation of human induced pluripotent stem cell derived retinal pigment epithelium (hiPSC-RPE), the determination of maturation status of these cells is an essential factor and the degree of pigmentation (dPG) can serve as a good indicator. The aim of this study was to establish a method of evaluating dPG in a group of hiPSC-RPE objectively and quantitatively.
Bright field images of hiPSC-RPE were recorded sequentially (0w, 2w, 4w, 6w after hiPSC-RPE reached confluence) under the same condition. Two observers, expert and inexpert, determined dPG subjectively by observation the recoded images as follows: a single cell dPG was classified into three different stages (low, mediate, high) and the overall dPG was compared between two cell groups to choose the one with higher dPG. The κ coefficient was applied to assess inter-observer reproducibility. Subsequently, dPG was objectively determined in single cells and cell groups by converting the recorded images into those with 256 gray-levels and the correlation with the subjective evaluation and time dependent change of dPG was investigated.
The κ coefficient was 0.96 and 0.87 in a single cell and cell group observation respectively, meaning that inter-observer reproducibility of dPG was excellent. The objective dPG as gray-level of single cells highly correlated with the subjectively determined dPG (low: 16.03, mediate: 71.11, high: 131.55, P < 0.001). However, in comparison between 2 cell groups, the subjective determination of high dPG cell groups was not a complete match with objective determination and sometimes the observers could not distinguish dPG between 2 cell groups (dPG subjective/objective: high/high 86%, high/low 3% undetermined 11%). The objective dPG in cell groups increased in a time-dependent manner (0w: 27.21, 2w: 51.85, 4w: 80.25, 6w: 90.03, P < 0.001).
The determination of dPG in cell groups of hiPSC-RPE using gray-scale image was reliable and excelled subjective difficulties in monitoring hiPSC-RPE maturation indicated by dPG in culture.
This PDF is available to Subscribers Only