title: Dopaminergic Neuronal Differentiation from the Forebrain-Derived Human Neural Stem Cells Induced in Cultures by Using a Combination of BMP-7 and Pramipexole with Growth Factors.
abstract: Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.
authors.affiliation: Department of Critical-Care Medicine, Qilu Hospital of Shandong University, Shandong UniversityJinan, China; Department of Psychiatry, College of Medicine, University of Illinois at ChicagoChicago, IL, USA.
authors.forename: HongNa
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Critical-Care Medicine, Qilu Hospital of Shandong University, Shandong University Jinan, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Psychiatry, College of Medicine, University of Illinois at Chicago Chicago, IL, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Research and Development, Cell and Tissue Bank of Shandong Province Jinan, China.
authors.forename: XiaoDun
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Research and Development, Cell and Tissue Bank of Shandong Province Jinan, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Anatomy, Capital Medical University Beijing, China.
authors.forename: WeiMing
authors.initials: W
authors.lastname: Duan
authors.affiliation: Department of Psychiatry, College of Medicine, University of Illinois at Chicago Chicago, IL, USA.
authors.forename: TingYu
authors.initials: T
authors.lastname: Qu
mesh_terms: D000704:Analysis of Variance; D052160:Benzothiazoles / Q000494:pharmacology*; D055419:Bone Morphogenetic Protein 7 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D004298:Dopamine / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000187:drug effects*; D004338:Drug Combinations; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000187:drug effects*; D000077487:Pramipexole; D016548:Prosencephalon / Q000166:cytology; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D055419:Bone Morphogenetic Protein 7; D018491:Dopamine Agonists; D050483:Dopamine Plasma Membrane Transport Proteins; D004338:Drug Combinations; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C493452:SLC18A2 protein, human; D050493:Vesicular Monoamine Transport Proteins; D000077487:Pramipexole; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine
keywords: BMP-7; PRX; TH; dopamine release; dopaminergic neurons; forebrain-derived human neural stem cells