Purchase this article with an account.
David M. Wu, Jennifer Aparicio, Analiese DiConti, Thomas C. Lee; Manipulation Of Gene Expression In Orbital Adipose-derived Mesenchymal Stem Cells From Retinoblastoma Patients Using Lentiviral Vectors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5894.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
There remains no accurate animal model for retinoblastoma due to unique differences between human and mouse retinal development. In this study, we explore the feasibility of modeling retinoblastoma using mesenchymal stem cells derived from retinoblastoma patients. Here we describe our efforts to transform orbital adipose tissue obtained during enucleation towards a retinal precursor state to serve as a model for the study of retinoblastoma.
Orbital adipose tissue samples were surgically harvested during enucleation of patient eyes with retinoblastoma. Tissue samples were digested with collagenase and then cultured in Mesenchymal Stem Cell (MSC) media (IMDM, 20% FBS, 1x ITS, 5 mM Glutamax). Adherent cells were further propagated in culture. A lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was used to introduce pluripotency factors. They were then introduced to a series of induction and differentiation media that have previously been used to direct human embryonic stem cells to a retinal progenitor cell identity. RNA was isolated from MSCs using the Qiagen RNeasy Mini Kit and reverse transcription was carried out using the iScript Reverse Transcription Kit (BioRad). PCR was then carried out using primers to eye-field transcription factors.
MSCs derived from orbital adipose tissue that were transfected with lentiviral vectors and then propagated in the above conditions were found to express the eye-field transcription factors Six3 and Six6. They did not express other eye-field transcription factors tested including lhx2, Rx, Chx10, and Pax6.
Mesenchymal stem cells transfected with lentiviral vectors containing the pluripotency factors Oct4, Sox2, Klf4, and c-Myc and grown under reprogramming and differentiation conditions can be induced to express a subset of the eyefield transcription factors. Further study will be necessary to understand the mechanism by which the MSCs are restricted from developing a more full retinal precursor state.
This PDF is available to Subscribers Only