Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells

Roy, Neeta S.; Nakano, Takahiro; Xuing, Li; Kang, Jian; Goldman, Steven A.

doi:10.1016/j.expneurol.2005.06.021

Cited by 145 publications

(111 citation statements)

References 42 publications

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“…Based on developmental principles, hESCs have been differentiated into neural lineage and different neuronal subtypes, including spinal motor neurons [16][17][18] and midbrain dopaminergic neurons [19][20][21]. Spinal motor neurons, which are hard to culture and maintain from primary tissues, can be generated in large quantity from ESCs.…”

Section: Introductionmentioning

confidence: 99%

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Wang

Zhang

2012

Cell Res

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Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-∆7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

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Section: Introductionmentioning

confidence: 99%

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Wang

Zhang

2012

Cell Res

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“…Beginning in 2001, several systems have been established to generate neural lineage from hESCs, which have provided a platform for the subsequent generation of neuronal subtypes 6,7 . Based upon developmental principles, several neuron types such as spinal motor neurons [8][9][10][11][12] , midbrain dopaminergic neurons [13][14][15] , and neural retinal cells 16,17 have been efficiently specified from human PSCs. This was done by applying critical morphogens which are important for the specification of these neuron types during in vivo development.…”

Section: Introductionmentioning

confidence: 99%

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Boisvert

Denton

et al. 2013

JoVE

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Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them. Video LinkThe video component of this article can be found at

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“…The potential of these cell lines can be further enhanced by genetic modifications, which may promote controlled differentiation of stem cells along a specific developmental pathway (3), facilitate purification of a certain type of cells in a mixed population of ES-derived cells (4), or correct genetic defects (5). In spite of all the potential this approach presents, the standard chemical or mechanical methods for transgene delivery exhibited very low efficiency in human ES cells, with the most effective approaches yielding only approximately 1 stably transfected cell per 10 5 cells (6,7).…”

Section: Introductionmentioning

confidence: 99%

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Xia

Zhang

Zieth

et al. 2007

Stem Cells and Development

151

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Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters-cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1α (EF1α)-in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.

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Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells

Cited by 145 publications

References 42 publications

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

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