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Wei Zhu, Yongzhen Miao, Haoyun Duan, Qingjun Zhou, Shen Wu, jingxue zhang, Ningli Wang, Kewei Wang; Defined- and Xeno-free differentiation of human iPSCs to functional trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5212.
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© ARVO (1962-2015); The Authors (2016-present)
We have successfully generated trabecular meshwork (TM) cells from human induced pluripotent stem cells (iPSCs) using the medium conditioned by the primary human TM cells. For future stem cell-based therapy for glaucoma, the production of functional TM cells from human iPSCs in a defined- and xeno-free condition is highly desirable. The goal of this study was to identify key factors critical for iPSCs differentiation, and validate a defined- and xeno-free protocol for generation of functional TM cells from human iPSCs.
Human iPSCs-derived TM cells after differentiation by conditioned medium for 1-3 months and primary TM cells were individually collected for RNA extractions and sequencing. AutoSome and GO analysis were performed to identify highly expressed receptors during differentiation, which were confirmed by qRT-PCR. A defined- and xeno-free differentiation protocol (MEM-alpha, 10% knockout serum, TGF-β1, NGF-β, erythropoietin, PGF2α, EGF) was optimized for induction of human iPSC differentiation. The similarity between the iPSC-derived cells and primary TM cells were determined by morphological observations, immunohistochemical staining with LAMA4, TIMP3, AQP1, Col IV antibodies, and the formation of cross-lined actin network (CLANs) after dexamethasone (DEX) treatment.
The expressions of receptors, HTGFBR, PTGER4, PTPRN, EDIL3 MEGF8, PTGFR were significantly higher in iPSC-derived TM cells than iPSCs. The primary TM cells from two individuals also exhibited high expression of these receptors. Defined- and xeno-free differentiated cells showed morphological resemble of human primary TM cells. Immunohistochemical staining demonstrated that these differentiated cells could robustly express the LAMA4, TIMP3, AQP1, and Col IV. The ratio of differentiated cells that formed CLANs was dramatically increased after DEX treatment (20.81% vs. 14.94%, p=0.0023), which was similar to DEX induced CLANs formation of the primary TM cells (19.69% vs. 13.52%, p=0.0001).
A defined- and xeno-free differentiation protocol is developed and optimized for generation of functional TM resembling cells from iPSCs in this study. The putative iPSC-derived TM cells express proteins characteristic for primary TM cells, and are capable of forming CLANs. Our protocol can also provide a guidance for Good Manufacturing Practice-production of TM cells for potential use in clinics.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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