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Vishal N. Rao, Aiguo Ni, Sai H. Chavala; Oct4, Sox2, Klf4 are Sufficient to Reprogram Ciliary Body Cells into Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):341.
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Atrophic age-related macular degeneration (AMD) is caused by retinal pigment epithelial (RPE) degeneration. Induced pluripotent stem (IPS) cells, essentially indistinguishable from embryonic stem (ES) cells, have been derived from skin fibroblasts using four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc. Fibroblast derived IPS cells have been differentiated into RPE cells and present a viable option to replace damaged RPE cells. Ciliary body (CB) cells may be a favorable cell source for IPS because they possess the same embryonic lineage as RPE cells. Since c-Myc is a known oncogene, reprogramming using only three factors is favorable for cell replacement therapy. The study focused on whether CB cells can be reprogrammed into IPS cells, and if three factors are sufficient for pluripotency.
The CB region of Sox2-GFP reporter mice eyes were dissected and cultured as a monolayer after enzymatic and mechanical trituration. Passage 2 CB cells were seeded onto mouse embryonic fibroblasts after lenti-viral transfection of Sox2, Klf4, Oct4, with and without c-myc to generate IPS. Resulting IPS cells were characterized by immunostaining and RT-PCR for embryonic stem cell markers. Embryoid bodies were formed by the hanging drop method, plated for differentiation, and characterized by RT-PCR to assess for differentiation into three germ layers. In vivo differentiation was assessed by teratoma formation after subcutaneous injection of IPS cells into immune deficient mice. IPS cells were injected into blastocysts to assess contribution in chimera mice.
Both three and four factor (3F and 4F) lenti-viral induction methods to form IPS cells demonstrated endogenous expression of Oct4, Sox2, Klf4, and c-Myc similar to mouse ES cells. 3F and 4F immunostaining were consistent with ES cell markers. In vitro and in vivo differentiation of IPS cells generated cells in all three germ layers.
We are the first group to demonstrate that CB cells can be reprogrammed into IPS, and that three factors are sufficient to generate IPS cells from the CB. Dispensing c-myc disposes of a known oncogene for safer cell transplantation. Future studies will determine if CB-IPS are more efficient than fibroblast-IPS for generating RPE cells.
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