Efficient Conversion of Astrocytes to Functional Midbrain Dopaminergic Neurons Using a Single Polycistronic Vector

Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e28719. DOI: 10.1371/journal.pone.0028719
Source: PubMed


Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat Parkinson's Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential utility in cell replacement therapies for PD or in modeling the disease in vitro, astrocyte-derived dopaminergic neurons offer the prospect of direct in vivo reprogramming within the brain. As a first step toward this goal, we report the reprogramming of astrocytes to dopaminergic neurons using three transcription factors – ASCL1, LMX1B, and NURR1 – delivered in a single polycistronic lentiviral vector. The process is efficient, with 18.261.5% of cells expressing markers of dopaminergic neurons after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.

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    • "Indeed, reactive astroglial cells isolated from non-neurogenic regions of the adult brain after local injury share hallmarks with NPCs and developmental radial glia (Sofroniew and Vinters, 2010), having the potential to be reprogrammed into induced neurons (Heinrich et al., 2010, 2011). Thus, forced expression of transcription factors are known to instruct neurogenesis in embryonic development, among which the basic-helix-loop-helix (bHLH) gene Neurogenin-2 (NEUROG2) or a combination of ASCL1, LMX1B, and NURR1 transcription factors direct reactive postnatal astrocytes in vitro toward generation of functional glutamatergic (Heinrich et al., 2011) or dopaminergic neurons (Addis et al., 2011), respectively. Furthermore, recent evidence demonstrates that the neuron-forming capacity of astrocytes is also active in vivo following neurodegeneration of the cortex and striatum (Guo et al., 2014; Magnusson et al., 2014; Niu et al., 2013; Torper et al., 2013), highlighting the existence of an endogenous cell source capable to restore connectivity and function following brain trauma. "
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    ABSTRACT: Recent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "the delivery of transcriptional factors Achaete-scute homolog 1 (ASCL1), LIM homeobox transcription factor 1-beta (LMX1B), and Nuclear receptor related 1 protein (NURR1) in a polycistronic lentiviral vector, primary mature mouse astrocytes can be reprogrammed into functional dopaminergic neurons (Addis et al., 2011). Combining these two studies, RGCs could be the intermediate cell type of the differentiation process from astrocyte to dopaminergic neurons, but future experiments are needed to test this hypothesis. "
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