Dopaminergic neuronal differentiation of neural stem cells in rats at different gestational ages

Chunlong KE; Baili CHEN; Huawei JIN; Chao YANG

ABSTRACT

BACKGROUND:

Differentiation inducing factors and gestational age influence the differentiation potential of embryonic neuralstem cells.

OBJECTIVE:

This study was designed to observe the differentiation potential of rat mesencephalic neural stem cells at differentgestational ages towards dopaminergic neurons.

DESIGN:

A randomized controlled observation.

SETTING:

Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou City, GuangdongProvince, China.MATERIALS This study was performed at the Laboratory of the First Affiliated Hospital of Sun Yat-sen University between Marchand September 2007. Thirty adult gestational SD rats, weighing 350 400 g, were provided by the Laboratory Animal Center of SunYat-sen University (Permission No. 2007-0034). The protocol was performed in accordance with ethical guidelines stated in Guide forthe use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources Commission on Life Sciences, National Research Council, China (1985). DMEM/F12 serum-free medium,B27 additives, epidermal growth factor, basic fibmblast growth factor, and fetal bovine serum (volume fraction0, 1) were purchased fromGibco Company, British; Interleukin lα, interleukin 11, and glial cell-derived neurotrophic factors were purchased from R&D Company,USA; In addition, leukaemia inhibitory factor (Perpotech, British), tyrosine hydroxylase(Santa Cruz, USA), nidogen antibody,microtubule-associated protein 2 antibody, and glial fibrillary acidic protein antibody(Chemicon, USA) were also used.

METHODS:

Six rats were randomly selected at each time point (on days 10,12,14,16, and 18 after gestation). After anesthesia, therats were sacrificed. Under the aseptic condition, fetal rat was harvested. Rat mesencephalic ventral brain tissue was isolated forculture of neural stem cells. Different gestational ages of rat brain-derived neural stem cells were separately cultured in theserum-free medium containing epidermal growth factors and basic fibroblast growth factors. After passage and amplification, theneural stem cells were induced to differentiate towards dopaminergic neurons in the medium containing interleukin lu, interleukin11, leukaemia inhibitory factors, glial cell-derived leukaemia inhibitory factors. On day 6 after induction and differentiation, thedopaminergic neurons were observed and identified by immunocytochemistry. After labeled by tyrosine hydroxylase, thedifferentiated dopaminergic neuron proportion was detected by a flow cytometer.MAIN OUTCOME

MEASURES:

The growth state of differentiated rat neural stem cells at different gestational ages and theimmunocytochemistry results. The tyrosine hydroxylase staining-positive neural stem cell proportion after induction anddifferentiation.

RESULTS:

Rat mesencephalic neural stem cell spheres on days 10,12, 14, 16, and 18 after gestation adhesively grew in thedifferentiation-inducing medium. The neural stem cells in the spheres gradually grew in radial tendency. On day 6 afterdifferentiation, most of the neural stem cells exhibited 1-2 long processes or several short processes. After nidogenimmunocytochemical staining, most of neural stem cells exhibited cytoplasm-positive. After culture for 6 days in the differentiationinducing medium, rat mesencephalic neural stem cells at gestational 10,12, 14, 16, and 18 days were detected by a flow cytometer.Results demonstrated that the proportion of tyrosine hydroxylase-positive cells was (10.3±2.5)%, (21.6±3.4)%, (16.7±2.8)%,(14.2±3.2)%, and (8.9±1.8)%, respectively. There was a significant difference in the proportion of tyrosine hydroxylase-positivecells among the cells at different gestational days (P < 0.05). Rat neural stem cells at gestational 12 days could be induced todifferentiate into dopaminergic neurons at the highest proportion.

CONCLUSION:

Mesencephalic neural stem cells of rats at different gestational days have different capabilities to differentiatetowards dopaminergic neurons. The proportion of dopaminergic neurons is the highest when mesencephalic neural stem cells ofrats at gestational 12 days.