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.

Hongna Yang,Heng Chen,Feng Wang,Weiming Duan,Jing Wang,Xiaodun Liu,Tingyu Qu

doi:10.3389/fncir.2016.00029

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.

Highlights

Parkinson disease (PD) as one of the most common neurodegenerative diseases is characterized by marked depletion of dopamine caused by dopaminergic neurons loss or degeneration in substantia nigra (SN) of the midbrain
Because BMP7 and PRX can upregulate the expression of DA neuron-associated key genes Nurr1 and Ngn2, and growth factors including acidic fibroblast factor, forskolin, and phorbol-12-myristae-13-acetate (TPA), have been confirmed in previous studies to enhance the expression of tyrosine hydroxylase (TH) in forebrain-derived Human neural stem cells (hNSCs) (Christophersen et al, 2006), we explored the possibility in the current studies to promote the differentiation of forebrain-derived hNSCs into DA-producing neurons by a combined application of PRX and bone morphogenetic proteins 7 (BMP-7) with these growth factors
Induction of Dopaminergic Differentiation hNSC spheres were mechanically dissociated into small spheres, part of which were plated on 12 mm glass coverslips coated with poly-l-lysine in 1 ml complete culture medium supplemented with 1% fetal bovine serum (FBS) for promoting cell attaching to the coverslips

Summary

Introduction

Parkinson disease (PD) as one of the most common neurodegenerative diseases is characterized by marked depletion of dopamine caused by dopaminergic neurons loss or degeneration in substantia nigra (SN) of the midbrain. Cell replacement therapy for PD, especially dopaminergic (DA) neurons, is considered to be the most promising candidate for restoring nigrostriatal DA transmission in the treatment of PD, Embryonic stem cells (ESCs) have been successfully induced to differentiate into DA neurons in vitro using various protocols (Kirkeby et al, 2012; Yang et al, 2014; Lim et al, 2015). Human neural stem cells (hNSCs) have been successfully isolated from fetal forebrains and these forebrain-derived hNSCs can be expanded in cultures for more than a year without losing their multipotency to differentiate into neurons and glial cells (Christophersen et al, 2006). Forebrain-derived hNSCs can serve as suitable cell sources to provide sufficient number of candidate cells and to differentiate into DA neurons for transplantation uses in PD. Forebrain-derived hNSCs appear to hardly differentiate into functional DA neurons, lacking the capacity to release dopamine, compared to midbrain-derived hNSCs, which limited their therapeutic application in PD

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Conclusion