Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons

Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Nature Communications (Impact Factor: 11.47). 07/2013; 4:2183. DOI: 10.1038/ncomms3183
Source: PubMed


Cell fate can be reprogrammed by modifying intrinsic and extrinsic cues. Here we show that two small molecules (forskolin and dorsomorphin) enable the transcription factor Neurogenin 2 (NGN2) to convert human fetal lung fibroblasts into cholinergic neurons with high purity (>90%) and efficiency (up to 99% of NGN2-expressing cells). The conversion is direct without passing through a proliferative progenitor state. These human induced cholinergic neurons (hiCN) show mature electrophysiological properties and exhibit motor neuron-like features, including morphology, gene expression and the formation of functional neuromuscular junctions. Inclusion of an additional transcription factor, SOX11, also efficiently converts postnatal and adult skin fibroblasts from healthy and diseased human patients to cholinergic neurons. Taken together, this study identifies a simple and highly efficient strategy for reprogramming human fibroblasts to subtype-specific neurons. These findings offer a unique venue for investigating the molecular mechanisms underlying cellular plasticity and human neurodegenerative diseases.

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    • "However , so far conversion of glial cells into neurons has been largely achieved using viral-based expression of transcription factors. In contrast, small molecules have been used to promote neural differentiation (Chambers et al., 2012), facilitate cell reprogramming (Ladewig et al., 2012; Li et al., 2014; Liu et al., 2013), or even directly reprogram fibroblasts into iPSCs (Hou et al., 2013), neuroprogenitor cells (NPCs) (Cheng et al., 2014), or neurons (Hu et al., 2015; Li et al., 2015). Compared to transcription-factorbased reprogramming, small molecules offer ease of use and a broader range of downstream applications. "
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    ABSTRACT: We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.
    Cell stem cell 10/2015; DOI:10.1016/j.stem.2015.09.012 · 22.27 Impact Factor
    • ") (Ladewig et al., 2012; Liu et al., 2013). Fibroblast identity was verified and the cells were lentivirally transduced to express rtTA and a 2A-peptide-linked transcript coding for Ngn2 and Ascl1, resulting in transgenic, but silent and expandable, AN fibroblasts (Figures 2B and S1). "
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    ABSTRACT: Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.
    Cell stem cell 10/2015; DOI:10.1016/j.stem.2015.09.001 · 22.27 Impact Factor
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    • ") showed that SoxC factors control the expression of neuron-specific structural proteins (e.g., b-tubulin III, DCX, Lis1) that are expressed by immature neurons of many, if not all, subtype lineages during adult and embryonic neurogenesis. Finally , SoxC proteins were shown to be required for neuronal reprogramming of somatic cells and when combined with bHLH transcription factors to enhance the efficiency of reprogramming of somatic cells into specific neuronal subtypes (Mu et al. 2012; Liu et al. 2013 "
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    ABSTRACT: Adult-generated dentate granule neurons have emerged as major contributors to hippocampal plasticity. Newneurons are generated fromneural stem cells through a complex sequence of proliferation, differentiation, and maturation steps. Development of the new neuron is dependent on the precise temporal activity of transcription factors, which coordinate the expression of stage-specific genetic programs. Here, we review current knowledge in transcription factor-mediated regulation of mammalian neural stem cells and neurogenesis and will discuss potential mechanisms of how transcription factor networks, on one hand, allow for precise execution of the developmental sequence and, on the other hand, allow for adaptation of the rate and timing of adult neurogenesis in response to complex stimuli. Understanding transcription factor-mediated control of neuronal development will provide new insights into the mechanisms underlying neurogenesis-dependent plasticity in health and disease. © 2015 Cold Spring Harbor Laboratory Press. All rights reserved.
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