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Sai H. Chavala, Aiguo Ni, Larysa Pevny; Oct 3/4 Alone Is Sufficient To Reprogram Ciliary Body Cells Into Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):276.
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Atrophic age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world, and is caused by retinal pigment epithelial (RPE) degeneration. Ciliary body (CB) cells are an attractive source for RPE regeneration because they originate from the same embryonic lineage as RPE cells and are surgically accessible. We have shown that CB cells can be reprogrammed into induced pluripotent stem (IPS) cells using exogenous Sox2, Klf4, and Oct 3/4 with and without c-Myc. Here, our goal was to reduce the genetic requirement further, and reprogram CB cells with only one factor (1F), Oct 3/4 alone; a feat that remains unattainable in fibroblasts. The purpose is to establish a safer IPS source for cell transplantation.
The CB region of wild-type and Sox2-EGFP reporter mice were dissected and cultured after enzymatic and mechanical trituration. Passage 2 CB cells were grown with and without sphere formation and then seeded onto mouse embryonic fibroblasts after lenti-viral transfection of Oct 3/4 with and without Klf-4 (2F) to generate IPS. Resulting IPS cells were characterized by immunostaining and RT-PCR for embryonic stem cell markers. Karyotyping also demonstrated normal chromosome numbers. Differentiation into three germ layers was assessed by in vivo teratomas after subcutaneous injection of IPS cells into immune deficient mice. IPS cells were injected into blastocysts and transplanted into pseduopregnant mice to assess IPS contribution in chimera mice.
Sphere formed CB cells can be reprogrammed into IPS using 1F or 2F unlike non-sphere formed CB cells. Sox2-EGFP reporter mice demonstrated that sphere formation increases Sox2 expression. Sphere formed CB cells from Sox2 deficient mice transfected with 1F did not generate IPS suggesting that SOX2 is essential for 1F IPS. 1F IPS differentiated into all three germ layers in teratomas from immune deficient mouse.
To date and to our knowledge, we are the first to reprogram a non-stem epithelial cell into IPS using Oct 3/4 alone. Sphere formation provides somatic CB cells with a reprogramming advantage over fibroblasts and other cell types by replacing exogenous SOX2. Reducing the genetic requirement necessary to generate IPS is ideal for safer human cell replacement therapy.
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