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      Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1

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          Summary

          Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

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          Highlights

          • ASCL1 alone generates functional neurons from fibroblast and embryonic stem cells
          • ASCL1-induced 1F-iN cells display slow maturation kinetics
          • ASCL1 overexpression induces endogenous expression of Myt1l and Brn2
          • ASCL1-induced 1F-iN cells are predominantly excitatory

          Abstract

          Direct reprogramming of nonneuronal cells into functional neurons is often considered to require overexpression of multiple transcription factors, but their functional hierarchy remained unknown. Here, Wernig, Südhof, and colleagues show that overexpression of an “on target” pioneer factor, ASCL1, is sufficient to generate induced neuronal cells from fibroblast and embryonic stem cells of both mouse and human origin.

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          Most cited references 25

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          Direct conversion of fibroblasts to functional neurons by defined factors

          Cellular differentiation and lineage commitment are considered 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, 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 modeling, and regenerative medicine.
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            Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming.

            Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. Here, we review strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming based on recent insights into the regulatory circuitry of the pluripotent state.
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              Rapid single-step induction of functional neurons from human pluripotent stem cells.

              Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening. Copyright © 2013 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                4 July 2014
                4 July 2014
                12 August 2014
                : 3
                : 2
                : 282-296
                Affiliations
                [1 ]Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology, Stanford University, Stanford, CA 94305, USA
                [2 ]Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA
                [3 ]Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
                [4 ]Department of Biology, Stanford University, Stanford, CA 94305, USA
                Author notes
                []Corresponding author tcs1@ 123456stanford.edu
                [∗∗ ]Corresponding author wernig@ 123456stanford.edu
                [5]

                Co-first author

                Article
                S2213-6711(14)00187-8
                10.1016/j.stemcr.2014.05.020
                4176533
                25254342
                © 2014 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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