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Anna Petelinsek, Elizabeth E Capowski, Sara Howden, Lynda S Wright, Isabel Pinilla Lozano, Kyle Wallace, Eric Clark, Joe Phillips, David M Gamm; Microphthalmia-associated Transcription Factor (MITF) affects optic vesicle cell proliferation and retinal pigment epithelium maturation in human ES cell (hESC) cultures. Invest. Ophthalmol. Vis. Sci. 2014;55(13):697.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of MITF expression results in microphthalmia and RPE defects in rodent models, but its role in human retinogenesis remains unknown. We created a MITF double knockout (dKO) hESC line to examine the consequences of its absence during early retinal development.
The second common exon of MITF was targeted by two rounds of BAC-mediated homologous recombination to inactivate both alleles in WA09 hESCs. Correct targeting and gene inactivation were confirmed by genomic PCR, RT-PCR, and Western blots. Light microscopy, immunocytochemistry (ICC), RT-PCR, and RT-qPCR were then performed at different stages of retinal cell development to examine the impact of MITF loss on the production, proliferation, and maturation of early neuroretinal cells and RPE.
RT-PCR and ICC analyses confirmed the absence of MITF transcripts and protein in dKO-MITF hESCs. Upon differentiation, ICC revealed that dKO-MITF hESCs sequentially adopted anterior neuroectoderm and early eye field fates in a manner similar to isogenic control WA09 hESCs. However, optic vesicle-like structures (OVs) generated from dKO-MITF hESCs were significantly smaller than control OVs (>60% reduction; p<0.0001). In addition, expression levels of PAX6, RX, and SIX6 were reduced in dKO-MITF vs. control OVs (54, 76, and 79% reductions, respectively), as was the number of proliferating cells (Ki67+ cells: 15.4% vs. 23.4%; p≤ 0.01). Following onset of VSX2 expression, cell proliferation within dKO-MITF OVs matched that of control OVs. Furthermore, dKO-MITF OVs produced CRX/Recoverin+ precursors in a temporal and spatial pattern indistinguishable from control OVs. However, RPE generated from dKO-MITF hESCs failed to fully mature; instead, dKO-MITF RPE remained unpigmented and lacked typical RPE organization even after several months in culture.
Using a genetically engineered hESC line, we showed that MITF is specifically involved in human OV proliferation and RPE maturation. Such information may lead to improvements in retinal cell production in vitro and/or provide insight into retinal development at stages previously inaccessible in humans.
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