A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells

Monzó, Héctor J.; Park, Thomas I-H; Montgomery, Johanna M.; Faull, Richard L.M.; Dragunow, Mike; Curtis, Maurice A.

doi:10.1016/j.jneumeth.2011.11.008

Cited by 28 publications

(22 citation statements)

References 68 publications

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“…P19 cells can be induced to differentiate into neuronal cells by retinoic acid, a process that closely resembles mammalian neurogenesis in vivo (Monzo et al, 2012). As reported earlier, MOR gene expression starts to increase within 2 days after the induction of differentiation, and continues further as the cells fully differentiate into neuronal subtypes (Hwang et al, 2010).…”

Section: Msk1 Expression Activates M-opioid Receptor Transcriptionsupporting

confidence: 53%

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1

Wagley

Law

et al. 2017

Mol Pharmacol

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Since the discovery of m-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogenactivated protein kinase (MAPK) activation and mitogen-and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

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Section: Msk1 Expression Activates M-opioid Receptor Transcriptionsupporting

confidence: 53%

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1

Wagley

Law

et al. 2017

Mol Pharmacol

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“…We used previously established monolayer cultures (Monzo et al 2012) for efficient morphological P19 cell differentiation by RA and showed that this process is driven by RARA by using RAR isoform-specific agonists (Supplemental Fig. S3A).…”

Section: Ra Induces Both Common and Cell Fatespecific Programs In F9 mentioning

confidence: 99%

Reconstructed cell fate–regulatory programs in stem cells reveal hierarchies and key factors of neurogenesis

Mendoza-Parra¹,

Malysheva²,

Saleem³

et al. 2016

Genome Res.

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Cell lineages, which shape the body architecture and specify cell functions, derive from the integration of a plethora of cell intrinsic and extrinsic signals. These signals trigger a multiplicity of decisions at several levels to modulate the activity of dynamic gene regulatory networks (GRNs), which ensure both general and cell-specific functions within a given lineage, thereby establishing cell fates. Significant knowledge about these events and the involved key drivers comes from homogeneous cell differentiation models. Even a single chemical trigger, such as the morphogen all-trans retinoic acid (RA), can induce the complex network of gene-regulatory decisions that matures a stem/precursor cell to a particular step within a given lineage. Here we have dissected the GRNs involved in the RA-induced neuronal or endodermal cell fate specification by integrating dynamic RXRA binding, chromatin accessibility, epigenetic promoter epigenetic status, and the transcriptional activity inferred from RNA polymerase II mapping and transcription profiling. Our data reveal how RA induces a network of transcription factors (TFs), which direct the temporal organization of cognate GRNs, thereby driving neuronal/endodermal cell fate specification. Modeling signal transduction propagation using the reconstructed GRNs indicated critical TFs for neuronal cell fate specification, which were confirmed by CRISPR/Cas9-mediated genome editing. Overall, this study demonstrates that a systems view of cell fate specification combined with computational signal transduction models provides the necessary insight in cellular plasticity for cell fate engineering. The present integrated approach can be used to monitor the in vitro capacity of (engineered) cells/tissues to establish cell lineages for regenerative medicine.

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“…al. (22) and has been demonstrated to generate a large number of fully mature neurons at both morphological and electrophysiological levels.…”

Section: Methodsmentioning

confidence: 99%

Murine Teratocarcinoma-Derived Neuronal Cultures

Datta¹

2013

Neuronal Cell Culture

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Summary This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19 and strategies for differentiation of these stem cells into neurons. Protocols focus on maintenance and propagation of these cells and routine procedures employed for differentiation into neuronal cells. Additional protocols are also described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be employed for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.

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A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells

Cited by 28 publications

References 68 publications

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1

Reconstructed cell fate–regulatory programs in stem cells reveal hierarchies and key factors of neurogenesis

Murine Teratocarcinoma-Derived Neuronal Cultures

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