Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine-lesioned parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.
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"Our experiments can be interpreted to demonstrate that a inhibition of Mef2C reduces promoter activity but does not rule out that other Mef2 family members may also contribute to the activity. Cho et al. report that forced expression of a constitutively active Mef2c increases the generation of neurons with dopaminergic properties derived from hESC-derived NPCs . These results agree with the hypothesis that the induction of miR-9-2 during neurogenesis is in part due to the activity of Mef2C, the only regulated isoform of Mef2 during neurogenesis. "
[Show abstract][Hide abstract]ABSTRACT: MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.
"It is now possible to routinely generate high numbers of ESC-derived neurons of various subtypes in vitro (Bibel et al., 2007; Parsons et al., 2011; Garcia et al., 2012; Kirkeby et al., 2012; Moon et al., 2012; Salti et al., 2012). Molecular marker analysis and electrophysiological characterizations have demonstrated that these neurons appropriately differentiate in vitro and exhibit patterns of action potentials and neurochemical profiles characteristic of neurons found in intact brain tissue (Bissonnette et al., 2011; Caiazzo et al., 2011; Cho et al., 2011; Garcia et al., 2012). Using specialized protocols, heterogeneous populations of NSCs, as well as GABAergic, glutamatergic, dopaminergic, and various other neuronal and glial subpopulations can be obtained at high quantities. "
[Show abstract][Hide abstract]ABSTRACT: The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons, now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs toward investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.
Full-text · Article · Dec 2012 · Frontiers in Cellular Neuroscience
"The hESC-derived NPCs had differentiated to the neuronal phenotype as evidenced by electrophysiological criteria, including firing action potentials (Fig. 1B) and immunocytochemical evidence based on multiple markers (Fig. 1D) (Li et al., 2008; Cho et al., 2011). MEF2CA-driven neuronal differentiation (Li et al., 2008; Cho et al., 2011) produced neurons with typical neuronal phenotype, as seen with immunocytochemical staining for the specific neuronal markers synapsin 1 and MAP2, synaptophysin, tau protein (Fig. 1D), and TuJ1 (Fig. 1E). When MEF2CA-programmed (compared with vector-infected control) hESC-derived neurons were analyzed for the presence of neurotransmitter specific markers, they showed high levels of expression of the synthetic enzymes tyrosine hydroxylase and glutamic acid decarboxylase (Fig. 1E), indicating an abundance of dopaminergic and GABAergic neurons, respectively. "
[Show abstract][Hide abstract]ABSTRACT: After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the β and γ ranges (10-100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.
Full-text · Article · Nov 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience