title: The colayer method as an efficient way to genetically modify mesencephalic progenitor cells transplanted into 6-OHDA rat model of Parkinson's disease.
abstract: Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However, the low survival rate of transplanted dopaminergic neurons (DA) calls for methodological improvements. Here we evaluated a method to combine transient genetic modification of neuronal progenitor  cells with an optimized cell culture protocol prior to intrastriatal transplantation into 6-hydroxydopamine (6-OHDA) unilateral lesioned rats. Plasmid-based delivery of brain-derived neurotrophic factor (BDNF) increases the number of DA neurons, identified by tyrosine hydroxylase immunoreactivity  (TH-ir), by 25% in vitro, compared to enhanced green fluorescence protein (EGFP)-transfected controls. However, the nucleofection itself, especially the cell detachment and reseeding procedure, decreases the TH-ir neuron number to 40% compared with nontransfected control cultures. To circumvent  this drawback we established the colayer method, which contains a mix of nucleofected cells reseeded on top of an adherent sister culture in a ratio 1:3. In this setup TH-ir neuron number remains high and could be further increased by 25% after BDNF transfection. Comparison of both cell culture  procedures (standard and colayer) after intrastriatal transplantation revealed a similar DA neuron survival as seen in vitro. Two weeks after grafting TH-ir neuron number was strongly reduced in animals receiving the standard EGFP-transfected cells (271 ± 62) compared to 1,723 ±  199 TH-ir neurons in the colayer group. In contrast to the in vitro results, no differences in the number of grafted TH-ir neurons were observed between BDNF, EGFP, and nontransfected colayer groups, neither 2 nor 13 weeks after transplantation. Likewise, amphetamine and apomorphine-induced  rotational behavior improved similarly over time in all groups. Nevertheless, the colayer protocol provides an efficient way for neurotrophic factor release by transplanted progenitor cells and will help to study the effects of candidate factors on survival and integration of transplanted  DA neurons.
authors.affiliation: Institute of Neuroanatomy, Hannover Medical School, Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ratzka
authors.forename: Ieva
authors.initials: I
authors.lastname: Kalve
authors.forename: Meltem
authors.initials: M
authors.lastname: Özer
authors.forename: André
authors.initials: A
authors.lastname: Nobre
authors.forename: Maike
authors.initials: M
authors.lastname: Wesemann
authors.forename: Julia
authors.initials: J
authors.lastname: Jungnickel
authors.forename: Christiane
authors.initials: C
authors.lastname: Köster-Patzlaff
authors.forename: Olga
authors.initials: O
authors.lastname: Baron
authors.forename: Claudia
authors.initials: C
authors.lastname: Grothe
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology*; D016627:Oxidopamine / Q000009:adverse effects*; D010300:Parkinson Disease / Q000628:therapy*; D051381:Rats; D017207:Rats, Sprague-Dawley; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016627:Oxidopamine