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Qiuli FU, Zhenwei QIN, Zhi FANG, Junfeng JI, Ke YAO; Generation of lentoid bodies from human urinary cell derived induced pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4010.
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© ARVO (1962-2015); The Authors (2016-present)
Cataract, opacification of lens, is a complex disease involving genetic and environmental factors, which is responsible for more than half of blindness worldwide. Lack of appropriate animal and cell models in vitro made it difficult to understand its pathological mechanisms, therefore limit the process of curing the disease. This study is aim to establish an in vitro system in which lens development and cataract process could be mimic using human induced pluripotent stem cells (hiPSCs).
Human exfoliated renal epithelial cells which present in urine were collected and cultured. Then, the isolated urinary cells were infected with four Yamanaka factors to generate urinary hiPSCs (UiPSCs) whose characteristics were examined by immunostaining and teratoma experiment. To establish a strategy to generate lentoid bodies (LBs) from UiPSCs, we adapted the 3-stage system of LBs formation from human embryonic stem cells (hESCs) (Yang et al, FASEB J, 2010). Expression of lens-specific markers was examined by real-time PCR, immunostaining and western blot. Structure of LBs was investigated using transmission electron microscropy (TEM).
Small colonies of urinary cells were observed at d4 and used for retroviruses infection after expansion. Three weeks after infection, UiPSCs were generated as evidenced by AP staining and expression of stem cells specific markers (NANOG, SSEA4, SOX2 and TRA1-81), as well as teratoma formation. 28 days after induction, LBs showing sphere-like shape and transparent structure were obtained (Fig.1) and expressed lens-specific markers including αA, αB, β and γ-crystallins, and MIP/aquaporin 0. More interestingly, transmission electron microscropy (TEM) revealed the presence of lens epithelial cells adjacent to the lens capsule, followed by unmature fiberlike cells with degenerating nucleus and organelles, and mature fiberlike cells without nucleus and organelles which located in the center of LBs. To further analyze whether LBs have the function of human lens, we examined their magnifying ability and approximately 1.7X of the magnification factor were found.
Our study established an in vitro system through an invasive procedure which can be utilized to studies of human lens development and cataractogenesis using patient specific UiPSCs.
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