Differentiation into dopaminergic neurons from neonatal rat neural stem cells induced by ascorbic acid*,*,☆

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Abstract

BACKGROUND:

Studies regarding cell transplantation for Parkinson's disease focuses on searching appropriate cell lines and induction of abundant functional dopaminergic (DA) neuron from cells with differentiation potential.

OBJECTIVE:

To investigate the effects of ascorbic acid (AA) on the differentiation of neural stem cells (NSCs) into DA neurons in vitro.

DESIGN, TIME AND SETTING:

In vitro observation of cytology. The experiment was performed at the Laboratory Center of Guangxi Medical University from January to December 2007.

MATERIALS:

A total of 30 Sprague Dawley rats (within 24 hours after birth) were provided by the Experimental Animal Center of Guangxi Medical University. AA was purchased from Sigma, USA.

METHODS:

The primary NSCs were isolated from whole brain of rats, suspended and cultured in serum-free medium in vitro at a density of 5×108/L (the second passage). Control cells were not given AA but only cultured in DMEM/F12 containing 10% fetal bovine serum and 2% B27; other cells were additionally induced with 50, 100, 200 μ mol/L for 10 days.

MAIN OUTCOME MEASURES:

Tyrosine hydroxylase (TH) mRNA was detected by RT-PCR; NSCs and differentiation from NSCs to DA neurons were detected by immunocytochemistry.

RESULTS:

Three AA groups had 795 bp amplified fragment, i.e. expressing TH mRNA. The neurospheres could proliferate and passage in vitro. Immunocytochemistry study indicated that the neurospheres were nestin-positive, had the capacity of self-renewing and proliferation, and could differentiate into multi-directions, specific antigens of neurons, astrocytes, and oligodendrocytes were expressed also. Compared with blank control group, AA increased the proportion of TH-positive cells (P < 0.05), in particular 100 and 200 μ mol/L was significantly higher than 50 μ mol/L (P < 0.05), while no significant difference was found between 100 and 200 μ mol/L AA (P > 0.05).

CONCLUSION:

The NSCs isolated from neonatal rats show the capacity of self-renewing and proliferation, multi-differentiation, and expressing nestin antigen in vitro. AA (50-200 μ mol/L) can promote NSCs differentiate into DA neurons, and TH positive rate minimally changes when AA increases from 100 to 200 μ mol/L.

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