Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury

Hamada, Mari; Yoshikawa, Hideshi; Ueda, Y.; Kurokawa, Manae S.; Watanabe, Kenji; Sakakibara, Manabu; Tadokoro, Mamoru; Akashi, Katsuya; Aoki, Haruhito; Suzuki, Noboru

doi:10.1016/j.nbd.2005.12.020

Cited by 40 publications

(35 citation statements)

References 61 publications

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“…12-18, normal karyotype) [14] . The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM), supplemented with 2 m M glutamine, 0.1 m M ␤ -mercaptoethanol, 1 !…”

Section: Mouse Escsmentioning

confidence: 98%

“…Total RNA extraction, cDNA synthesis and polymerase chain reaction (PCR) amplification have been reported [14] . ␤ -Actin was used to detect housekeeping gene expression in all reversetranscriptase (RT)-PCRs.…”

Section: Reverse-transcriptase Polymerase Chain Reactionmentioning

confidence: 99%

“…Intracellular free calcium levels were evaluated using fluo-3-acetoxymethyl (AM) ester (Dojindo, Kumamoto, Japan) [14,22,23] . Briefly, 14 days after switching DMEM/10% FCS medium to DMEM/F12 medium with N2 supplement and fibronectin, more than 90% of the cloned pax6 -transfected cells were Brn3 positive on immunostaining ( fig.…”

Section: Calcium Imagingmentioning

confidence: 99%

“…To see the voltage-gated calcium influx, 100 m M KCl was added after recording the base emission intensity. Ca 2+ influx was induced by adding KCl with a pipette onto the cells under investigation as well as to proximal cells from 0.8 to 2 s. It has been shown under a whole-cell current clamp that the application of 100 m M KCl led to a constant membrane potential of -10 mV [14,24] .…”

Section: Calcium Imagingmentioning

confidence: 99%

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Transfection with <i>pax6</i> Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

et al. 2009

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Objective: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. Methods: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. Results: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing βIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca²⁺ imaging. Conclusions: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

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“…12-18, normal karyotype) [14] . The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM), supplemented with 2 m M glutamine, 0.1 m M ␤ -mercaptoethanol, 1 !…”

Section: Mouse Escsmentioning

confidence: 98%

Section: Reverse-transcriptase Polymerase Chain Reactionmentioning

confidence: 99%

Section: Calcium Imagingmentioning

confidence: 99%

Section: Calcium Imagingmentioning

confidence: 99%

See 2 more Smart Citations

Transfection with <i>pax6</i> Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

et al. 2009

Self Cite

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“…The balance of these factors plays an important role in neural differentiation in vivo, [41], and it is possible that a change in the bHLH expression pattern can regulate neural differentiation, laying a foundation for the treatment of disease by cell transplantation. For instance, Mash1 protein promotes the generation of neural precursor cells and the neural differentiation of multipotent stem cells [42]. The expression of neurogenin2 (ngn2) in neural progenitor cells of the central and peripheral nervous systems can accelerate the differentiation of these progenitor cells into neurons [43,44].…”

Section: Neural Differentiation and Immunocytochemistrymentioning

confidence: 99%

The effect ofId1gene silencing on the neural differentiation of MSCs

Song

et al. 2017

Biotechnology & Biotechnological Equipment

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We aimed to construct a highly efficient, lentivirus-mediated vector for RNA interference (RNAi) silencing of the Id1 gene and to examine whether silencing of this gene promotes the neural differentiation of mesenchymal stem cells (MSCs). Three short hairpin RNA sequences targeting the coding region of the Id1 gene and one negative control (NC) sequence were designed. After monophosphorylation, these sequences were introduced into lentiviral vectors containing enhanced green fluorescent protein (EGFP) using the restriction enzyme BsmBI to construct the recombinant vectors pWSLV-EGF-shId1 (1-3) and pWSLV-EGF-NC. These recombinant lentivirus vectors were transfected into H9C2 cells to assess the silencing effect of the shId1 (1-3) sequences on the Id1 gene. Subsequently, pWSLV-EGF-NC and pWSLV-EGF-shId1 which exhibited the strongest silencing effect were transfected into the packaging cell line 293 FT. After determining the viral titers, the cultured MSCs were infected with the lentivirus (multiplicity of infection, MOI D 10). Real-time polymerase chain reaction (PCR) and Western blotting were used to examine the amounts of Id1 mRNA and protein, respectively, in the transfected MSCs, and the results revealed that Id1 expression was markedly inhibited. Finally, MSCs were treated with the growth factors EGF and bFGF in conjunction with ligustrazine to induce their differentiation into neuron-like cells. The expression of NSE/MAP-2/Nestin was detected using immunocytochemistry and real-time PCR, which revealed that the rate of neural differentiation in the pWSLV-EGF-shId1 group was higher than that of the other groups, as was NSE/MAP-2 expression. Thus, Id1 gene silencing promotes the neural differentiation of MSCs.

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Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

Furmanski

Gajavelli

Lee

et al. 2009

J of Comparative Neurology

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Numerous CNS disorders share a common pathology in dysregulation of γ-amino butyric acid inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated FGF-2 deprivation and MASH1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and β-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased BrdU incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased β-III-tubulin and decreased CNPase expression, while FGF-2 deprivation alone attenuated GFAP expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

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Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury

Cited by 40 publications

References 61 publications

Transfection with <i>pax6</i> Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

Transfection with <i>pax6</i> Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

The effect ofId1gene silencing on the neural differentiation of MSCs

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

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