Generation of Dopaminergic Neurons in the Adult Brain from Mesencephalic Precursor Cells Labeled with anestin-GFPTransgene

Abstract

Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein (GFP) cDNA under the control of the nestin enhancer. nestin-driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP ϩ ) cells. After sorting by fluorescence-activated cell sorting, the GFP ϩ cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP-sorted mesencephalic precursors developed a dopaminergic phenotype in vitro. Finally, nestin-GFP ϩ cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine-tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.

Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons

“…The cells were isolated under fluorescence microscopy. The isolated cells were suspended in 1 ml of DMEM-F12 containing B-27 with 1% methylcellulose (Sigma-Aldrich), and 20 ng⅐ml Ϫ1 basic FGF (bFGF) (Chemicon) (5,14). Cells were cultured in 24-well tissue-culture dishes (Corning) at 37°C in a 5% CO 2 ͞95% air tissue-culture incubator.…”

“…The cells were resuspended into fresh RPMI medium 1640 (Cellgro, Herndon, VA) containing 10% FBS. The colonies were cultured in SonicSeal four-well chamber slides (Nunc) (5,14).…”

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

“…Cryosections and cultured cells were prepared for immunofluorescence analyses as described (14). Mouse anti-TH mAb (17) diluted by 1:500 or rabbit anti-TH antibody (18) diluted by 1:10,000 was used as the primary antibody in combination with a Cy3-conjugated secondary antibody (Jackson ImmunoResearch), diluted by 1:1,000.…”

“…FACS sorting of GFP ϩ cells was performed on a FACS Vantage flow cytometer͞cell sorter (Becton Dickinson), essentially as described (13)(14)(15)(16). Dead cells were excluded by gating on forward and side scatter and by elimination of cells stained with propidium iodide, a fluorescent dye labeling only dead cells.…”

A transgenic mouse Class‐III β tubulin reporter using yellow fluorescent protein

“…Nestin-GFP mice have been used in studying neural stem and progenitor cells in the developing and adult nervous systems and those GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain (Kawaguchi et al, 2001;Mignone et al, 2004;Sawamoto et al, 2001) . In this paper we present β-tubulin III -YFP transgenic lines that offer similar promise as experimental tools to mark the conversion of neuronal precursor cell into fully differentiated neurons (Jiang and Oblinger, 1992), the acquisition of populations of differentiating neurons in developing and adult tissues using cell sorting methods, and the prelabeling of specific populations of cells for in vitro analysis to transplant into the adult or developing CNS to examine the behavior of differentiating neurons in a novel environment.…”