Neuronal Development of Embryonic Stem Cells: A Model of GABAergic Neuron Differentiation

doi:10.1006/bbrc.2001.5031

Biochemical and Biophysical Research Communications

Volume 284, Issue 3, 15 June 2001, Pages 674-680

https://doi.org/10.1006/bbrc.2001.5031 Get rights and content

Abstract

Neural cultures derived from differentiating embryonic stem (ES) cells are a potentially powerful in vitro model of neural development. We show that neural cells derived from mouse ES cells express mRNAs characteristic of GABAergic neurons. The glutamate decarboxylase genes (Gad1 and Gad2), required for GABA synthesis and the vesicular inhibitory amino acid transporter (Viaat) gene, required for GABA vesicular packaging are activated in the ES-derived cultures. Nearly half of the ES-derived neurons express the GAD67 protein, the product of the Gad1 gene. Building on these results we show that Gad1-lacZ “knockin” reporter ES cell lines can be used to easily monitor Gad1 expression patterns and expression levels during ES differentiation. We also demonstrate that the ES-derived neural progenitors can be infected with retroviruses or transfected with plasmids via lipofection. These experiments outline the basic strategies and methods required for studies of GABAergic gene expression and regulation in ES-derived neuronal cultures.

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Cited by (56)

The Nervous System
2013, Principles of Tissue Engineering: Fourth Edition
The central nervous system (CNS) has been proposed as one of the prime targets for pluripotent stem (PS) cell-based therapies, due to early successes in directing PS cell towards neural fates, due to the experience with fetal tissue transplantation in neurodegenerative diseases, and due to the devastating nature of many CNS diseases which often lack efficient alternative treatments. Some of the most striking advantages of embryonic stem cells as compared with any other cell type are: extensive self-renewal capacity and differentiation potential, access to the earliest stages of neural development, and ease of inducing stable genetic manipulations. The development of protocols that allow the directed differentiation from embryonic stem (ES) and induced pluripotent stem (IPS) cells to specific neural fates provides an essential basis for all PS cell-based approaches in neural repair. Over the last few years, directed differentiation protocols have rapidly progressed and neural induction is now a routine procedure for studies in both mouse and human PS cells.
Over-expression of Mash1 improves the GABAergic differentiation of bone marrow mesenchymal stem cells in vitro
2013, Brain Research Bulletin
Citation Excerpt :
The expression of both the neuronal (NeuN, β3-tubulin) and GABAergic (GAD67, GABA) markers was increased in the presence of Mash1 overexpression in the differentiated BMSCs cultures. GABA, the major inhibitory neurotransmitter in the nervous system, synthesized by the GAD65/67 enzymes, expressed in GABAergic neurons (Westmoreland et al., 2001). We investigated the immunoreactivity of this marker and found a higher proportion of GABA-positive neurons in the Mash1-overexpressed differentiated BMSCs compared to controls.
Bone marrow mesenchymal stem cells (BMSCs) have been shown to be a promising cell type for the study of neuronal differentiation; however, few attempts had been made to differentiate these cells into inhibitory gamma-aminobutyric acid (GABA)ergic neurons. In this study, we over-expressed mammalian achaete-scute homologue-1 (Mash1), a basic helix-loop-helix (bHLH) transcription factor, in Sprague-Dawley rat BMSCs via lentiviral vectors, and then induced neuronal differentiation of these cells using conditioned medium. Our Western blot results show that, under conditions of differentiation, Mash1-overexpressing BMSCs exhibit an increased expression of neuronal markers and a greater degree of neuronal morphology compared to control, non-Mash1-overexpressing cells. Using immunocytochemistry, we observed increased expression of glutamic acid decarboxylase 67 (GAD67), as well as neuron-specific nuclear protein (NeuN) and β3-tubulin, in Mash1-overexpressing BMSCs compared to control cells. Moreover, we also found the differentiated cells showed representative traces of action potentials in electrophysiological characterization. In conclusion, our study demonstrated that over-expression of Mash1 can improve GABAergic differentiation of BMSCs in vitro.
Modeling Huntington's disease with induced pluripotent stem cells
2013, Molecular and Cellular Neuroscience
Citation Excerpt :
After EB generation, morphogens such as RA are added to induce neural patterning (Bain et al., 1995), after which cells are plated to induce GABAergic specification (Strubing et al., 1995). Similar to the EB method, the monolayer protocol utilizes patterning morphogens, such as FGF, in defined concentrations and for specific periods of time to induce GABAergic specification (Westmoreland et al., 2001). This methodology is thought to generate neural cells more efficiently than other methods as the media and conditions are more defined and likely more closely recapitulate neural development (Abranches et al., 2009).
Huntington's disease (HD) causes severe motor dysfunction, behavioral abnormalities, cognitive impairment and death. Investigations into its molecular pathology have primarily relied on murine tissues; however, the recent discovery of induced pluripotent stem cells (iPSCs) has opened new possibilities to model neurodegenerative disease using cells derived directly from patients, and therefore may provide a human-cell-based platform for unique insights into the pathogenesis of HD. Here, we will examine the practical implementation of iPSCs to study HD, such as approaches to differentiate embryonic stem cells (ESCs) or iPSCs into medium spiny neurons, the cell type most susceptible in HD. We will explore the HD-related phenotypes identified in iPSCs and ESCs and review how brain development and neurogenesis may actually be altered early, before the onset of HD symptoms, which could inform the search for drugs that delay disease onset. Finally, we will speculate on the exciting possibility that ESCs or iPSCs might be used as therapeutics to restore or replace dying neurons in HD brains.
Epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from mouse embryonic stem cells
2013, Journal of Biological Chemistry
The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.
Background: Orexin plays a central role in the integration of sleep/wake states and feeding behaviors.
Results: Orexin neurons were induced from pluripotent stem cells by supplementation of ManNAc.
Conclusion: ManNAc induced switching of epigenetic factors from Sirt1/Ogt to Mgea5 at Hcrt gene locus.
Significance: This study will be useful to investigate molecular mechanism in the orexin system and development of regenerative medicine.
The Nervous System
2013, Handbook of Stem Cells
Exogenous GDNF increase the migration of the neural stem cells with no protection against kainic acid-induced excitotoxic cell death in rats
2012, Brain Research
Citation Excerpt :
The overexpressed GDNF induces genes regulating the migration and differentiation of neural progenitor cells (Jens et al., 2004). These stem cell-derived neurons express the GAD-67 protein or GABA, thereby identifying them as GABAergic neurons (Sabine et al., 2005; Xiaoxiao et al., 2005; Joby et al., 2001; Hiroki et al., 2000; Jonathan et al., 1998). Therefore, the enhanced GAD-67 expression observed in the Ad-GDNF-NSCs-treated rats is believed to be attributable to the differentiation of NSCs, a process which is facilitated by the overexpressed GDNF (Fig. 4J–L).
Glia cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuron types. In this study, we have evaluated the utility of adenovirus-based vectors (Ad) and hippocampal neural stem cells (NSCs) as genetic tools for the delivery of a therapeutic protein, GDNF, in hippocampus tissues damaged by kainic acid (KA)-induced excitotoxicity. The experimental animals were treated with KA 3 days prior to exposure to Ad-GDNF, NSCs, and NSCs infected with Ad-GDNF (Ad-GDNF-NSCs). Seven days after the treatments with Ad-GDNF, NSCs and Ad-GDNF-NSCs, the effects of the treatments were evaluated. GAD-67 labeled cells originating from the transplanted NSCs were observed at increased levels in the Ad-GDNF-NSCs-treated rats as compared to the NSCs-only rats. In situ apoptosis assays showed that the levels of TUNEL-positive cells were slightly, but not significantly, reduced in the Ad-GDNF and Ad-GDNF-NSCs groups, as compared to the saline and NSCs only groups. GDNF expression by NSCs and Ad-GDNF was upregulated as the consequence of adenoviral gene delivery in the NSCs and Ad-GDNF-treated rats, and the transplanted NSCs were shown to have migrated to the hippocampal regions in Ad-GDNF-NSCs rats to a greater degree than in the NSCs-only rats. Furthermore, in the region in which the NSCs were detected, GDNF and GAD-67 expression were increased. These results indicate that the migration and differentiation of NSCs may be associated with the expression of GDNF. However, cell death consequent to KA administration was not prevented by upregulated GDNF and NSCs transplantation. Collectively, our results indicate that GDNF may exert effects on the migration and differentiation of NSCs, but there are no protective properties with regard to excitotoxically damaged hippocampal tissue.

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¹: To whom correspondence should be addressed at Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, CB 2803, Augusta, GA 30912. Fax: 706-721-8685. E-mail: [email protected].

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Biochemical and Biophysical Research Communications

Abstract

References (24)

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells

Dev. Biol.

Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro

Mech. Dev.

Two genes encode distinct glutamate decarboxylases

Neuron

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases

FEBS Lett.

Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control

Cell

Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells

Biochem. Biophys. Res. Commun.

Site directed mutagenesis by gene targeting in mouse embryo derived stem cells

Cell

Embryonic stem cells express neuronal properties in vitro

Dev. Biol.

Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons

Mech. Dev.

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

Cell Transplant

Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons

Mech. Dev.

Cited by (56)

The Nervous System

Over-expression of Mash1 improves the GABAergic differentiation of bone marrow mesenchymal stem cells in vitro

Modeling Huntington's disease with induced pluripotent stem cells

Epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from mouse embryonic stem cells

The Nervous System

Exogenous GDNF increase the migration of the neural stem cells with no protection against kainic acid-induced excitotoxic cell death in rats

Regular ArticleNeuronal Development of Embryonic Stem Cells: A Model of GABAergic Neuron Differentiation

Abstract

Dev. Biol.

Mech. Dev.

Neuron

FEBS Lett.

Cell

Biochem. Biophys. Res. Commun.

Cell

Dev. Biol.

Mech. Dev.

Cell Transplant

Mech. Dev.

Regular Article
Neuronal Development of Embryonic Stem Cells: A Model of GABAergic Neuron Differentiation