title: Nucleofection is the most efficient nonviral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate.
abstract: Neuronal progenitor cells (NPCs) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, such as Parkinson disease. However, survival of transplanted cells is, as yet, limited, and the identification of grafted cells in situ remains difficult. The use of NPCs could be more effective with regard to a better survival and maturation when transfected with one or more neurotrophic factors. Therefore, we investigated the possibility of transfecting mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters enhanced green fluorescence protein (EGFP) and red fluorescent protein (DsRed). Different techniques for transfection were compared, and the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure; 6 hours after transfection, viability was approximately 40%, and the transfected cells differentiated into, for example, tyrosine hydroxylase-positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. To provide the progenitor cells with a neurotrophic factor, different isoforms of fibroblast growth factor-2 were introduced. To follow the behavior of the transfected cells in vitro, functional tests such as the cell viability assay (water-soluble tetrazolium salt assay [WST-1]) and the cell proliferation assay (5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay) were performed. In addition, these transfected NPCs were viable after transplantation, expressed tyrosine hydroxylase in vivo, and could easily be detected within the host striatum because of their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases.
authors.affiliation: Department of Neuroanatomy, Center for Systems Neuroscience, Hannover, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Cesnulevicius
authors.forename: Marco
authors.initials: M
authors.lastname: Timmer
authors.forename: Maike
authors.initials: M
authors.lastname: Wesemann
authors.forename: Tobias
authors.initials: T
authors.lastname: Thomas
authors.forename: Tanja
authors.initials: T
authors.lastname: Barkhausen
authors.forename: Claudia
authors.initials: C
authors.lastname: Grothe
mesh_terms: D000818:Animals; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D004298:Dopamine / Q000378:metabolism*; D018274:Electroporation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology* / Q000473:pathology / Q000637:transplantation; D009474:Neurons / Q000166:cytology*; D016627:Oxidopamine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection / Q000379:methods*; D014780:Viruses
chemical_list: C485184:enhanced green fluorescent protein; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D016627:Oxidopamine; D004298:Dopamine