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D. E. Nelidova, C.-Y. Chang, J. McGhee, T. Sherwin, C. R. Green; In situ Reprogramming of Keratocytes Into Neurons. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3765.
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© ARVO (1962-2015); The Authors (2016-present)
Cell reprogramming induces changes in the gene expression of adult cells, allowing them to change phenotype. Reprogramming commonly involves reversion to an intermediate pluripotent cell type and usually relies on gene transfection. Identifying the molecules that drive phenotypic changes circumvents the need for gene therapy, and performing reprogramming in situ enhances therapeutic possibilities.
An organotypic air-liquid interphase culture of adult human corneal tissue slices was established using proneural Neurobasal -A complex supplemented with Epidermal Growth Factor (EGF) and Fibroblast Growth Factor-2 (FGF-2) or serum containing control media. Expression of neural progenitor and mature neuronal markers was assessed using immunohistochemistry.
After three days in proneural media, keratocytes en masse expressed Musashi-1 and Nestin, indicating acquisition of neural precursor properties. There was no evidence for morphologically immature cells being involved. After one week, the cells now expressed Nestin, beta III tubulin, SMI-32, MAP-2 and Neurofilament-200, and had developed a neuronal morphology, indicating progression towards a mature neuronal phenotype. Reprogramming occurred across the entire thickness of the corneal stroma, although new neurons remained spatially constrained between collagen layers. Both EGF and FGF-2 were essential for reprogramming, removal of either prevented transdifferentiation.
We demonstrate for the first time that en masse cellular reprogramming of adult fibroblastic cells to neurons in situ is feasible. Transdifferentiation was induced directly, not via an intermediate stem or pluripotent cell phenotype, and did not involve gene transfer. We suggest the evidence for tissue-specific stem cells arising in embryogenesis and persisting into adulthood may need to be reassessed when all adult fibroblasic cells are amenable to reprogramming. This finding has relevance to corneal nerve replacement and other tissue engineering.
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