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Elizabeth Capowski, Joseph Simonett, Eric Clark, Lynda Wright, Isabel Pinilla Lozano, Joe Phillips, Kyle Wallace, David Gamm; MITF regulation of early cell fate in a human embryonic stem cell model of retinal development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4049.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the effect of MITF on early retinal cell fate and maturation by manipulating its expression in differentiating human embryonic stem cells (hESCs).
Protocols for hESC culture, targeted retinal differentiation, immunocytochemistry, and RT-qPCR have been described ( Phillips et al. (2012) IOVS 53:2007). Stable hESC lines expressing short hairpin RNA (shRNA) were generated by infecting WA09 hESCs with lentiviral vectors, selecting with blasticidin, and monitoring for GFP expression. For ectopic VSX2 expression, lentivirus was used to transduce hESC cultures during early stages of retinal differentiation.
Early optic vesicle-like (OV) cell fate was identified in hESCs in part via expression of MITF in a subset of PAX6+ cells after approximately 2 weeks of differentiation. Over the next three days, VSX2 was co-expressed with MITF and subsequently solely expressed in mounded neuroretinal OV clusters, which could be detached by gentle trituration and cultured separately. Following their removal, MITF expression was retained in the surrounding skirt of epithelial-like cells which, over the course of the next three weeks, differentiated into RPE. Stable subclones of hESCs expressing pan-MITF shRNA reduced MITF gene expression by 65% compared to control shRNA, and also reduced relative levels of eye field, but not forebrain, markers. Later during differentiation, when RPE fate is typically well-established, expression of MITF remained reduced in the pan-MITF shRNA line, as did that of the MITF targets BEST1 and TYR. However, expression levels of the RPE markers MERTK, RPE65, and CRALBP were unaltered. When VSX2 was ectopically expressed in early MITF+ epithelia, MITF expression was significantly reduced and RPE generation was suppressed. In contrast, neuroretina and forebrain fates were unaffected.
This data suggests that in human, as in other mammals, MITF plays a role in OV development as well as RPE pigmentation, and that VSX2 can repress MITF expression and thus also affect OV fate.
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