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Danni Lyu, Qiuli Fu, Ke Yao; Characteristics analyses of CRYBB2 and CRYGD-mutated congenital cataract patients' iPSCs-derived lentoid bodies in vitro. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1204.
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© ARVO (1962-2015); The Authors (2016-present)
The pathological mechanisms underlying congenital cataract remain largely unknown due to the lack of appropriate in vitro cellular models. The present study aims at uncovering the aberrant progress of lens generation in gene mutation-related congenital cataract by means of an in vitro model of human lens regeneration using induced pluripotent stem cells (iPSCs).
Urinary cells were collected from healthy individuals and congenital cataract patients with CRYBB2 (Q155X) or CRYGD (P24T) mutation and infected with four Yamanaka factors to generate urinary cells-derived iPSCs (UiPSCs). UiPSCs were then induced to differentiate into lentoid bodies (LBs) using our previously established "fried egg" method. The morphology of UiPSCs-derived LBs was examined by light microscope. The expressions of lens-specific markers were examined by real-time PCR and immunofluorescence staining. The cellular arrangement was analyzed by histochemical staining. The soluble and insoluble proteins were extracted and quantified by modified Lowery assay, respectively.
The CRYBB2-mutated UiPSCs-derived LBs (CRYBB2-mutated LBs) had similar progress of generation with those from healthy individuals and developed opacity in the late stage (Day 21), while the differentiation of CRYGD-mutated UiPSCs-derived LBs (CRYGD-mutated LBs) were relatively retarded with earlier occurrence of opacity (Day 17) (see Figure 1). Real-time PCR and immunofluorescence staining revealed similar expressions of the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin between healthy and mutated LBs, whereas the expression of β-crystallin was significantly decreased in CRYBB2-mutated LBs (p<0.01), and so as γ-crystallin in CRYGD-mutated LBs (p<0.01). Methylene blue staining showed irregular dense cord-like structures in the middle of mutated LBs. The ratio of insoluble/soluble proteins of CRYBB-mutated and CRYGD-mutated LBs were 1.97 and 2.39 times the ratio of normal LBs, respectively (p<0.01).
The present study simulated and analyzed the process of lens development and cataractogenesis in vitro by LBs differentiation from congenital cataract patients-derived UiPSCs, which set up a platform for pathological mechanisms research and clinical drug screening of congenital cataract.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Images of mature LBs after 25 days of differentiation.
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