Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line—Studies in vitro and after intracerebral grafting in vivo

Paul, Gesine; Christophersen, Nicolaj S.; Raymon, Heather K.; Kiaer, Caroline; Smith, Ruben; Brundin, Patrik

doi:10.1016/j.mcn.2006.11.010

Cited by 32 publications

(30 citation statements)

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“…Addition of tetracycline in combination with neurotrophic mediators enables an exit from the cell cycle and differentiation into a neuronal phenotype (Lotharius et al, 2005). LUHMES are a single cell subclone of MESC2.10 cells that were originally generated as an alternative source of graft material for transplantations in Parkinson's disease (PD) patients (Hansson et al, 2000;Lotharius et al, 2002;Paul et al, 2007). Based on their mesencephalic origin, lUHMeS then were introduced as an in vitro PD model (Lotharius et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Schildknecht

Karreman

Pöltl

et al. 2013

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Summary -phenylpyridinium (MPP + ). Different lentiviral constructs then were tested: cells labeled ubiquitously with green (GFP) or red fluorescent protein (RFP) allowed the quantification of neurite growth and of its disturbance by toxicants; expression of proteins of interest could be targeted to different organelles; production of two different proteins from a single read-through construct was achieved successfully by an expression strategy using a linker peptide between the two proteins, which is cleaved by deubiquitinases; LUHMES, labeled with GFP in the cytosol and RFP in the mitochondria

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Section: Introductionmentioning

confidence: 99%

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Schildknecht

Karreman

Pöltl

et al. 2013

ALTEX

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“…Alternative strategies based on the suppression of the oncogene used to immortalize primary cells have been described, such as the use of inducible promoters (Hoshimaru et al 1996;Lotharius et al 2002;Paul et al 2007), which can be manipulated thereby allowing the cells to differentiate. Another approach involves the modification of the oncogene itself.…”

Section: Discussionmentioning

confidence: 99%

“…After 24 h, the adhesion medium was replaced with proliferation medium (a modification of adhesion medium without FCS and B27 supplement) or by differentiation medium (DMEM/F12, 0.25% [w/v] BSA, B27, 1% FCS, 100 μM ascorbic acid, 2 mM glutamine). To test the composition of the differentiation medium, 1 mM di-butyryl cyclic adenosine monophosphate (cAMP, Sigma-Aldrich) and 2 ng/ml glial cell line-derived neurotrophic factor (GDNF, Tebu-bio) were added to enhance differentiation (Paul et al 2007). …”

Section: Cell Culturementioning

confidence: 99%

“…With regard to the production of immortalized cells derived from the ventral midbrain, a conditionally human immortalized mesencephalic cell line has been established taking advantage of a tetracycline-regulated gene expression system, thereby allowing the constitutive expression of v-myc in the absence of tetracycline (Hoshimaru et al 1996;Lotharius et al 2002). Under differentiating culture conditions, these cells display neuronal electrical activity and the expression of the rate-limiting enzyme of DA synthesis, viz., tyrosine hydroxylase (TH; Lotharius et al 2002;Paul et al 2007). However, intracerebral grafting of these cells has revealed no TH expression (Paul et al 2007).…”

mentioning

confidence: 99%

“…Under differentiating culture conditions, these cells display neuronal electrical activity and the expression of the rate-limiting enzyme of DA synthesis, viz., tyrosine hydroxylase (TH; Lotharius et al 2002;Paul et al 2007). However, intracerebral grafting of these cells has revealed no TH expression (Paul et al 2007). In addition, immortalized mesencephalic cells derived from rat embryos have been generated by transfection with a plasmid vector expressing SV40Tag (Prasad et al 1994).…”

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Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen

et al. 2010

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Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.

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Cell Transplantation and Gene Therapy in Parkinson's Disease

Wakeman

Dodiya

Kordower

2011

Mount Sinai J Medicine

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Parkinson's disease is a progressive neurodegenerative disorder affecting, in part, dopaminergic motor neurons of the ventral midbrain and their terminal projections that course to the striatum. Symptomatic strategies focused on dopamine replacement have proven effective at remediating some motor symptoms during the course of disease but ultimately fail to deliver long-term disease modification and lose effectiveness due to the emergence of side effects. Several strategies have been experimentally tested as alternatives for Parkinson's disease, including direct cell replacement and gene transfer through viral vectors. Cellular transplantation of dopamine-secreting cells was hypothesized as a substitute for pharmacotherapy to directly provide dopamine, whereas gene therapy has primarily focused on restoration of dopamine synthesis or neuroprotection and restoration of spared host dopaminergic circuitry through trophic factors as a means to enhance sustained controlled dopamine transmission. This seems now to have been verified in numerous studies in rodents and nonhuman primates, which have shown that grafts of fetal dopamine neurons or gene transfer through viral vector delivery can lead to improvements in biochemical and behavioral indices of dopamine deficiency. However, in clinical studies, the improvements in parkinsonism have been rather modest and variable and have been plagued by graft-induced dyskinesias. New developments in stem-cell transplantation and induced patient-derived cells have opened the doors for the advancement of cell-based therapeutics. In addition, viral-vector-derived therapies have been developed preclinically with excellent safety and efficacy profiles, showing promise in clinical trials thus far. Further progress and optimization of these therapies will be necessary to ensure safety and efficacy before widespread clinical use is deemed appropriate.

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Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line—Studies in vitro and after intracerebral grafting in vivo

Cited by 32 publications

References 44 publications

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen

Cell Transplantation and Gene Therapy in Parkinson's Disease

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