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Direct conversion of fibroblasts to functional neurons by defined factors

Abstract

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

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Figure 1: A screen for neuronal-fate-inducing factors and characterization of MEF-derived iN cells
Figure 2: Efficient induction of neurons from perinatal tail-tip fibroblasts
Figure 3: The 5F-pool-induced conversion is rapid and efficient
Figure 4: MEF-derived iN cells show functional synaptic properties.
Figure 5: Defining a minimal pool for efficient induction of functional iN cells

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Acknowledgements

We would like to thank S. Marro and P. Lovelace for help with FACS sorting, S. Hafeez and Y. Huh for assistance with molecular cloning and mouse husbandry, and K. Jann for assistance with the diagram in Fig. 1. We would also like to thank I. Graef, R. Bajpai, J. Wysocka, J.-R. Lin and J.-Y. Chen for contributing reagents and help with analysis. This work was supported by start-up funds from the Institute for Stem Cell Biology and Regenerative Medicine at Stanford (M.W.), the Donald E. and Delia B. Baxter Foundation (M.W.), an award from William Stinehart Jr and the Reed Foundation (M.W.), the National Institute of Health Training Grant 1018438-142-PABCA (A.O.) and the Ruth and Robert Halperin Stanford Graduate Fellowship (T.V.). Z.P.P. is supported by NARSAD Young Investigator Award and NIH/NINDS Epilepsy Training Grant 5T32NS007280.

Author Contributions T.V., A.O. and M.W. designed and conceived the experiments. T.V., Y.K. and M.W. produced the lentiviral vectors. T.V. and A.O. performed the lentiviral infections, isolated the fibroblasts and completed the molecular characterization of the iN cells. Z.P.P. and T.C.S. designed, performed and analysed the electrophysiological assays. T.V., A.O., Z.P.P., T.C.S. and M.W. wrote and edited the manuscript and produced the figures.

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Correspondence to Marius Wernig.

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Vierbuchen, T., Ostermeier, A., Pang, Z. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010). https://doi.org/10.1038/nature08797

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