Combined use of BDNF, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells.
Freshly isolated fetal midbrain neural precursor cells (NPCs) that maintain the potential to differentiate into dopamine (DA) neurons represent a valuable source for cell therapy in Parkinson's disease. However, it is poorly understood why midbrain NPCs lose their dopaminergic differentiation potential after long-term culture. Here we report that human fetal midbrain NPCs can be extensively proliferated with fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) and efficiently differentiated into tyrosine hydroxylase-immunoreactive (TH-ir) neurons. We tested differentiation conditions including the use of low oxygen, ascorbic acid, and prolonged in vitro differentiation time which resulted in a 10-fold increase in the number of MAP2-positive neurons (up to 40-50% of total cells as compared to controls). Under these conditions TH-ir cells constituted 4.3+/-0.5% of the neuronal population and displayed immature morphologies. Notably, the use of brain-derived neurotrophic factor (BDNF) further increased the proportion of TH-ir neurons (up to 15% of total neurons). In contrast to previous reports, our findings demonstrate that long-term expanded fetal NPCs can generate TH-expressing cells under the appropriate culture conditions and without genetic manipulations.
Maciaczyk
Jaroslaw
J
Laboratory of Molecular Neurosurgery, Department of Stereotactic and Functional Neurosurgery, Neurocenter, University of Freiburg, Freiburg, Germany.
Singec
Ilyas
I
Maciaczyk
Donata
D
Nikkhah
Guido
G
D001205:Ascorbic Acid / Q000008:administration & dosage*; D019208:Brain-Derived Neurotrophic Factor / Q000008:administration & dosage*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005333:Fetus; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008636:Mesencephalon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013995:Time; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis* / Q000235:genetics
D019208:Brain-Derived Neurotrophic Factor; D014446:Tyrosine 3-Monooxygenase; D001205:Ascorbic Acid; D010100:Oxygen