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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Available from: Meng-Lu Liu
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    • "Small molecules inducing iPSCs can be classified into three types: (1) small molecules that improve reprogramming efficiency[55]; (2) compounds replacing one or more reprogramming factors565758; and (3) compound cocktails alone that are sufficient to induce iPSCs[59,60]. Small molecule methods have been successfully applied to reprogram mouse fibroblasts directly into functional neurons using only a combination of small molecules[59,60]. Induced neural progenitor cells (iNPCs) have also been generated using a chemical cocktail comprised of an inhibitor of GSK-3 kinases and TGF-β pathways under physiologically hypoxic conditions[61]. "

    Full-text · Article · Jan 2016 · International Journal of Molecular Sciences
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    • "Primary fibroblasts (Table S1) from three normal (NL) healthy adult humans (AG05811, 71 years, designated NL1; AG07473, 50 years, designated NL2; and AG09969, 53 years, designated NL3) were co-transduced with lentiviruses expressing NEUROG2-IRES-GFP-T2A-SOX11 and ISL1-T2A- LHX3 (hereafter referred to as NSIL). Then, 2 days post-viral infection (dpi), these cells were switched to neuron-induction media containing our previously identified extrinsic factors, forskolin (FSK) and dorsomorphin (DM), and basic fibroblast growth factor (FGF2) (Liu et al., 2013). Neuronal conversion was monitored daily by live-cell fluorescence microscopy and analyzed by immunocytochemistry at the indicated time points. "
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    ABSTRACT: Subtype-specific neurons obtained from adult humans will be critical to modeling neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Here, we show that adult human skin fibroblasts can be directly and efficiently converted into highly pure motor neurons without passing through an induced pluripotent stem cell stage. These adult human induced motor neurons (hiMNs) exhibit the cytological and electrophysiological features of spinal motor neurons and form functional neuromuscular junctions (NMJs) with skeletal muscles. Importantly, hiMNs converted from ALS patient fibroblasts show disease-specific degeneration manifested through poor survival, soma shrinkage, hypoactivity, and an inability to form NMJs. A chemical screen revealed that the degenerative features of ALS hiMNs can be remarkably rescued by the small molecule kenpaullone. Taken together, our results define a direct and efficient strategy to obtain disease-relevant neuronal subtypes from adult human patients and reveal their promising value in disease modeling and drug identification.
    Full-text · Article · Dec 2015 · Cell Reports
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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.
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