Optimization of ultrastructure observation of human embryonic stem cell-derived retinal pigment epithelial cells

Authors: Wu Wei,  Qu Ya,  Wang Lei,  Zeng Yuxiao,  Xu Haiwei,  Yin Zhengqin

 DOI: 10.3760/cma.j.issn.2095-0160.2016.09.004
Published 2016-09-10
Cite as Chin J Exp Ophthalmol, 2016,34(9): 786-790.

Abstract

Background

Normal ultrastructure is the anatomical basis of retinal pigment epithelial(RPE)cells to perform normal physiological function.At present the precipitation method is often used to detect the ultrastructure of RPE cells with transmission electron microscopy(TEM).

Objective

The aim of this study was to explore a simple and feasible approach to examine the ultrastructure of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells.

Methods

hESCs were induced and differentiated into RPE cells by the spontaneous differentiation method, and the expressions of microphthalmia associated transcription factor MITF and paired-box gene 6 (PAX6), specific protein of RPE cells, in the cells were detected by immunofluorescence assay.hESC-RPE cells were inoculated into Transwell filter, and the ultrastructure of the cell sheet was examined under the TEM.Then the ultrastructure of the cell sheet specimens was compared with those of hESC-RPE cells from cell precipitation and RPE cell specimens of 90-day-old Long Evans rats.

Results

MITF and PAX6 were positively expressed in hESC-RPE cells.The normal ultrastructure were visible in the RPE cells of rats under the TEM, including apical microvilli, polarized melanin granules, cellular nucleus, basement membrane and intercellular junctions, and the ultrastructure of hESC-RPE cell sheet on Transwell was similar to the RPE cells in rats.However, only scatter melanin granules, nonpolar nucleus and scanty microvilli were observed under the TEM in the hESC-RPE cells by cell precipitation method.

Conclusions

Without digestion process, hESC-RPE cell sheet on Transwell can retain the normal ultrastructure of hESC-RPE cells under the TEM, with a more simple and reliable advantage.

Key words:

Retinal pigment epithelium/cytology; Embryonic stem cells; Microscopy, electron, transmission; Cell differentiation/physiology; Cells, cultured; Rats; Retinal pigment epithelial sheets, human

Contributor Information

Wu Wei
Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Qu Ya
Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Wang Lei
State Key of Stem Cells and Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
Zeng Yuxiao
Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Xu Haiwei
Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Yin Zhengqin
Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
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