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

ABSTRACT 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, Sep 29, 2015
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    • "When expressed in astrocytes obtained from postnatal murine cerebral cortex gray matter , Ascl1 instructs GABAergic neurons, while Neurog2 elicits glutamatergic neurons (Berninger et al., 2007; Heinrich et al., 2010), thus making possible the identification of target genes involved in neuronal subtype specification within the same transcriptional background. In different cell types, such as fibroblasts, Ascl1 induces a glutamatergic neuronal fate in combination with Myt1L and Brn2 in fibroblasts (Vierbuchen et al., 2010), while Neurog2 forces motor neuron generation in combination with forskolin and dorsomorphin (Liu et al., 2013). Thus, the cell of origin, with its specific epigenetic landscape, can play a role in defining the spectrum of reprogramming possibilities. "
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    ABSTRACT: Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "Bypassing pluripotency and directly reprogramming readily accessible human tissues, such as skin, into neural cells may offer a fast and efficient approach to study neurological disorders (Caiazzo et al., 2011; Pang et al., 2011; Yoo et al., 2011). Although direct neuronal conversion may offer unique benefits, this approach is currently limited to a small number of protocols to specify neuronal subtypes using postnatal or adult human samples (Caiazzo et al., 2011; Liu et al., 2013; Ring et al., 2012; Son et al., 2011; Yoo et al., 2011). MiR-9/9* and miR-124 are critical components of a genetic pathway that controls the assembly of neuron-specific, ATPdependent chromatin remodeling complexes during neural development (Staahl et al., 2013; Yoo et al., 2009). "
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    ABSTRACT: The promise of using reprogrammed human neurons for disease modeling and regenerative medicine relies on the ability to induce patient-derived neurons with high efficiency and subtype specificity. We have previously shown that ectopic expression of brain-enriched microRNAs (miRNAs), miR-9/9(∗) and miR-124 (miR-9/9(∗)-124), promoted direct conversion of human fibroblasts into neurons. Here we show that coexpression of miR-9/9(∗)-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, and MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of neurons analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, the reprogrammed human cells persisted in situ for over 6 months, exhibited membrane properties equivalent to native MSNs, and extended projections to the anatomical targets of MSNs. These findings highlight the potential of exploiting the synergism between miR-9/9(∗)-124 and transcription factors to generate specific neuronal subtypes.
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    • "Subtype specific iNs can be obtained from human fibroblasts5678, and thus represent a supply of human neurons that can be generated on demand and used in biomedical applications and for disease modelling. For example, induced neurons obtained via direct conversion of fibroblasts from a patient with Alzheimer's disease or from transgenic mice with autism-associated neuroligin-3 mutation have been shown to mimic pathologic conditions and abnormal neuronal phenotype910, substantiating their predicted use as disease models for neurological disorders as a quicker and simpler alternative to iPS cells for this purpose. "
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    ABSTRACT: Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.
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