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Binbin Xie, Xiang-Mei Zhang, Takao Hashimoto, Amy Tien, Andrew Chen, Jian Ge, Xian-Jie Yang; Differentiation of Retinal Ganglion Cells from Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2240.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal projection neurons are permanently damaged in disease conditions such as glaucoma and optic nerve neurophathy, One strategy for future retinal repair is to develop stem cell based therapies. Induced pluripotent stem cells (iPSCs) can be derived from patient somatic tissues and have the potential to differentiate into multiple cell lineages. The purpose of this study is to define and optimize conditions under which RGCs can differentiate efficiently from iPSCs.
Embryonic fibroblasts from transgenic mice encoding Cre from the Atoh7 locus and R26R.YFP reporter were obtained at E13.5. MEFs were induced towards pluripotent state using viral mediated expression of the Yamanaka reprogramming factors. The resulting iPSCs were validated using stem cell markers by immunocytochemistry and RT-PCRs. Selected iPSC lines were allowed to form embryoid bodies and developed towards the neural retina lineages. At various time of differentiation, retinal progenitor and neuronal cell markers were characterized using by immunohistochemistry and confocal imaging, western blot, and fluorescence activated cell sorting.
The Atoh7.YFP iPSCs show typical embryonic stem cell (ESC) morphology and can be passaged for many generations in standard ESC medium with LIF. In addition, Atoh7.YFP iPSCs express alkaline phosphatase, as well as Nanog and Oct4. Under conditions promoting the anterior neural fate, iPSC-derived cultures express retinal progenitor markers, including Rx, Pax6, and Chx10. After further differentiation, Atoh7 iPS cultures contain post mitotic neurons expressing Islet1, Pax6, Brn3a, neurofilaments, and β-Tubulin. Importantly, Brn3 and YFP reporter double positive neurons emerge from these cultures with extensive neurites, suggesting that these cells were derived from Atoh7 knockin Cre expressing progenitors. Further analyses by RT-PCR, Western blots, and immunolabeling have confirmed the identity of postmitotic RGCs derived from Atoh7 iPSCs.
Mouse iPSCs encoding the Atoh7.YFP reporter have been established and successfully passaged. The Atoh7.YFP iPSCs can differentiate in vitro and express Atoh7 gene and the YFP reporter. Neuronal marker analyses indicate that retinal neurons including RGCs are derived from Atoh7.YFP iPSCs. The YFP reporter can be used to optimize RGC differentiation and monitor transplanted RGCs.
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