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Lin Cheng, Qiannan Lei, Kangxin Jin, Mengqing Xiang; An optimized non-integrative protocol generates urine cell-derived human iPS cells for the study of genetic eye diseases. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3755.
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© ARVO (1962-2015); The Authors (2016-present)
Using a practical approach to generate iPS cells (UiPSCs) from human urine cells (UCs) will enable us to establish ocular disease models from a convenient source. In this report, we optimized a non-integrative protocol to generate patient-specific iPSC lines with high reprogramming efficacy, and established Brn3b-GFP knockin UiPSC lines via CRISPR/Cas9 technique for studying disease-causing mutations.
The iPSC reprogramming protocol was optimized from a previous report (Li, et al. 2016). 1.5×106 UCs were electroporated with 6ug pEP4-E02S-ET2K and 4ug pEP4-M2L plasmid DNAs [day(d)0], and cultured with RE/MC medium in matrigel-coated 6-well plate. At d1 and d2, the medium was changed to RE/MC medium plus 0.5uM A-8301, PD0325901, Thiazovivin and 3uM CHIR99021 (4i). At d3, it was changed to N2B27 medium +4i till the clones upheaved, when the medium was replaced with N2B27 medium +0.5uM Thiazovivin. Morphology changes were seen at d4-7. After d10, the clones were picked when the clone diameter >1mm. They were then further expanded and characterized by qPCR, embryoid body formation, AP staining, karyotyping, and labeling with pluripotency markers. The generated iPSCs were cultured in 3D to generate optic cups (Ohlemacher, et al. 2016). Moreover, CRISPR/Cas9-engineered UiPSCs were generated (Ran, et al. 2013).
UiPSCs were successfully derived from UCs with ~80 clones/well. They showed typical hESC morphology and were self-renewable. They maintained normal karyotype and are AP-positive. PCR assay confirmed that these iPSC colonies had no genomic integration. qPCR, flow cytometry and cell fluorescence immunostaining all confirmed that UiPSCs displayed high expression of pluripotency markers Sox2, Nanog, Oct4, SSEA-4, Tra-1-60 and Tra-1-81. UiPSCs were capable of differentiating into three germ layers. In 3D culture, UiPSCs formed retinal cups with laminar layers expressing BRN3A, BRN3B, TUJ1, NF150, MATH5, CRX and other retinal cell markers. A RGC reporter Brn3b-GFP UiPSC cell line was successfully established to investigate RGC degenerative diseases in the future.
The optimized protocol is an easy and fast approach to yield good-quality iPSC lines. The generated iPSCs are able to generate retinal cups, and the CRISPR/Cas9-engineered UiPSCs will allow us to establish patient-specific iPSCs, and manipulate genes to better understand ocular diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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