Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells

Abstract

Citation: Fu Q, Qin Z, Jin X, et al. Generation of functional lentoid bodies from human induced pluripotent stem cells derived from urinary cells. Invest Ophthalmol Vis Sci. 2017;58:517-527. DOI:10.1167/ iovs.16-20504 PURPOSE. The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells. METHODS.Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter ''X,'' respectively. RESULTS.We developed a ''fried egg'' differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers aA-, aB-, b-, and c-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, aA-, and aB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.73) of the LBs generated from UiPSCs. CONCLUSIONS.Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

A New Long Noncoding RNA ALB Regulates Autophagy by Enhancing the Transformation of LC3BI to LC3BII during Human Lens Development

“…Western blot analysis revealed the expression of LC3BII in mature LBs in our previous study. 24 Thus, the expression patterns of LC3B and P62 at different time points during LB differentiation as well as the location of LC3B were examined. Our results showed that more LC3B and less P62 were expressed in differentiating LBs after D14 when massive lens epithelial cells differentiated to fiber cells with organelle degradation ( Figures 2 A–2D).…”

“…Our previous study established an efficient system—a “fried egg” method for human lens regeneration in vitro from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). 24 The lens generated in the dish (also known as the lentoid body [LB]), which showed a lens-like transparent structure, not only expressed lens-specific markers but also exhibited basic optical characteristics. More importantly, in addition to lens-specific markers, including α, β, γ-crystallins, and MIP, the expression of placodal markers, such as SIX1 and PAX6, and early lens-specific markers, such as SOX1 and PROX1, were also observed at certain time points of LB differentiation.…”

A Human Three-Dimensional In Vitro Model of Lens Epithelial Cells as a Model to Study Mechanisms of Drug-Induced Posterior Subcapsular Cataracts

“…Celojevic et al 29 were also not able to detect E-cadherin expression by immunohistochemistry and by western blot in human lens epithelial cells derived from cataract surgery in patients. On the contrary, Fu et al 10 were able to detect E-cadherin in lentoid bodies made from humaninduced pluripotent stem cells derived from urinary cells.…”

“…5,6 Several lens in vitro models have been explored in the past including however not limited to primary and immortalized cell lines, 7,8 whole lens culture, 9 and lentoid bodies. 10 Although the benefit of cell line-derived models is the reproducibility and availability of cells, 11 whole lens cultures isolated from different species like rats 9 or mice 12 offer a great structural similarity to in vivo models. However, translation to human from animal lens explants seems to be challenging.…”

“…11 Only one human relevant model generated from human-induced pluripotent stem cells derived from urinary cells creating lentoid bodies in vitro has recently shown functional and structural characteristics, which are comparable with the human lens. 10 The aim of this study was to develop a human relevant lens in vitro model to investigate mechanisms of druginduced cataracts. The first aim was to establish a straightforward and reproducible model of the human lens in vitro.…”