338 IN VITRO GENERATION OF LENTOID BODIES FROM INDUCED PLURIPOTENT STEM CELLS OF TRANSGENIC MICE

Abstract

Pluripotent cells have the developmental potential to generate all adult cell types, so ocular diseases resulting from the failure of specific cell types could be potentially treatable through the transplantation of differentiated cells derived from stem cells. The present study was conducted with the aim of generating a cataract model. We attempted to derive the induced pluripotent stem (iPS) cells from fibroblast cells of transgenic (crytom) mice carrying a transgenic construct-alphaA crystallin promoter driving the tandem dimer (td) Tomato marker transgene, integrated in the genome. The 4- to 6-week-old female crytom mice were selected, superovulated, and mated. The fetuses were recovered and examined on various different days (10.5 to 15.5 days postfertilization), and the reporter expression was found to be initiated 12.5 days postfertilization and the intensity was increased thereafter. The expression of tdTomato was confirmed in the fetuses by Western blotting. Murine embryonic fibroblast (MEF) cultures were generated and electroporated with a reprogramming transposon cassette carrying Yamanaka factors (OCT4, SOX2, KLF4, and MYC) and Sleeping Beauty transposase to generate iPS cells which were picked up and clonally expanded. The cells were confirmed by PCR for tdTomato in the genome and characterised for the expression of Oct4 and cryAB by immunofluorescence. The iPS cells were also injected into the nude CD1 mice to test for teratoma formation. The generated cells were allowed to differentiate spontaneously on 3 different types (viz. P19, NTERA, and STO) of cell lines as feeders, in the absence of LIF, and cells were expected to fluoresce if differentiated to eye lens lineage. After long-term cultures, the iPS cells were found to differentiate and form lentoid bodies which expressed tdTomato. Thus, alphaA crystallin-tdTomato construct was allowed following lens cell formation by specific fluorescence excitation in a spatial and temporal manner. The employment of cell type-specific reporters for establishing and optimizing targeted differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations. Hence a transgenic murine iPS cell line was generated which exhibited potential to be used as a model for eye cataracts and other eye abnormalities.