Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE.
OBJECTIVE
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 κ 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 κ 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.
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Hyogo, Japan.
Hiroyuki
H
Kamao
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Hyogo, Japan.
Michiko
M
Mandai
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Shunji
S
Wakamiya
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Junko
J
Ishida
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Katsutoshi
K
Goto
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Takaaki
T
Ono
Tissue Biology & Electron Microscopy Research Center, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Taiji
T
Suda
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Hyogo, Japan.
Masayo
M
Takahashi
Department of Ophthalmology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Junichi
J
Kiryu
D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D009414:Nerve Growth Factors / Q000378:metabolism; D015588:Observer Variation; D010858:Pigmentation; D015203:Reproducibility of Results; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor
degree of pigmentation; human induced pluripotent stem cell; retinal pigment epithelium