Expansion and characterization of ventral mesencephalic precursor cells: effect of mitogens and investigation of FA1 as a potential dopaminergic marker.
Methods for identification and in vitro expansion of ventral mesencephalic dopaminergic precursor cells are of interest in the search for transplantable neurons for cell therapy in Parkinson's disease (PD). We investigated the potential use of fibroblast growth factor 2 (FGF2) and fibroblast growth factor 8 (FGF8) for expansion of such dopaminergic precursor cells, and fetal antigen-1 (FA1), a secreted neuronal protein of unknown function, as a non-invasive dopaminergic marker. Tissue from embryonic day (ED) 12 rat ventral mesencephalon was dissociated mechanically and cultured for 4 days in the presence of FGF2, FGF8, or without mitogens (control). After mitogen withdrawal and addition of 0.5% bovine serum, cells were differentiated for 6 days. Before differentiation, significantly more cells incorporated BrdU in cultures exposed to FGF2 (19-fold; P < 0.001) and FGF8 (3-fold; P < 0.05) compared to controls. After differentiation, biochemical analyses showed significantly more dopamine and FA1 in conditioned medium from both FGF2 and FGF8 expanded cultures than in controls. Correspondingly, numbers of tyrosine hydroxylase (TH)- and FA1-immunoreactive cells had increased 16-fold (P < 0.001) and 2.1-fold (P < 0.001), respectively in the FGF2 group and 10-fold (P < 0.001) and 1.8-fold (P < 0.05), respectively in the FGF8 group. In conclusion, the present procedure allows efficient expansion and differentiation of dopaminergic precursor cells and provides novel evidence of FGF8 as a mitogen for these cells. Furthermore, FA1 was identified as a potential supplementary non-invasive marker of cultured dopaminergic neurons.
Jensen
Pia
P
Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark.
Bauer
Matthias
M
Jensen
Charlotte H
CH
Widmer
Hans R
HR
Gramsbergen
Jan B
JB
Blaabjerg
Morten
M
Zimmer
Jens
J
Meyer
Morten
M
D000818:Animals; D000963:Antimetabolites; D015415:Biomarkers / Q000032:analysis; D001973:Bromodeoxyuridine; D002452:Cell Count; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004298:Dopamine / Q000378:metabolism / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D051524:Fibroblast Growth Factor 8 / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008636:Mesencephalon / Q000166:cytology*; D008934:Mitogens / Q000494:pharmacology*; D011247:Pregnancy; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D013234:Stem Cells / Q000502:physiology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000963:Antimetabolites; D015415:Biomarkers; D017077:Culture Media, Conditioned; C496458:Fgf8 protein, rat; D008934:Mitogens; D016222:Fibroblast Growth Factor 2; D051524:Fibroblast Growth Factor 8; D012701:Serotonin; D005680:gamma-Aminobutyric Acid; D014446:Tyrosine 3-Monooxygenase; D001973:Bromodeoxyuridine; D004298:Dopamine