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.

Fibroblast growth factor 23 signaling in hippocampal cells: impact on neuronal morphology and synaptic density

“…Cells were transfected on DIV5 with pCAGGS‐enhanced green fluorescent protein (EGFP)‐FLAG plasmid kindly provided by Dr Andreas Ratzka (Ratzka et al . ). The CAG promoter is composed of both, the cytomegalovirus and chicken β‐actin promoter and allows for high protein expression specifically in neurons (Niwa et al .…”

Cooperation of Nuclear Fibroblast Growth Factor Receptor 1 and Nurr1 Offers New Interactive Mechanism in Postmitotic Development of Mesencephalic Dopaminergic Neurons

“…Nurr1 expressing pCAGGSNurr1-FLAG plasmid was generated from pCAGGS-empty provided by Dr. Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) (21), which comprised the chicken ␤-actin promoter with CMV enhancer (CAG) promoter composed of CMV immediate early enhancer and chicken ␤-actin promoter. The generation of pCAGGS-3XFLAG plasmid was described previously (22). The full-length murine Nurr1 coding sequence was PCR-amplified from embryonic mouse brain cDNA; thereby an MfeI site followed by an Kozak sequence was introduced in front of the start codon.…”

Anodal Transcranial Direct Current Stimulation Enhances Survival and Integration of Dopaminergic Cell Transplants in a Rat Parkinson Model

“…Evidence for its trophic effects on fetal cell survival and differentiation in vitro is undisputed ( Hyman et al, 1991 ; Knüsel et al, 1991 ; Studer et al, 1995 ). In vivo , effects of BDNF on fiber outgrowth and behavior have also been promising ( Sauer et al, 1993 ; Yurek et al, 1998, 1996 ), despite methodological challenges, including the route of administration due to the large molecule size ( Yan et al, 1994 ; Krobert et al, 1997 ; Ratzka et al, 2012 ). Additionally, nonphysiologic continuous BDNF overexpression and trkB signaling may even have deleterious effects, such as seizures ( Croll et al, 1999 ; Heinrich et al, 2011 ).…”