Differentiation of neural stem cells derived from human fetal brain into neurons in young rat brains / 中国组织工程研究

ABSTRACT

AIM: Site-specific functional neurons of brains were with different cellular morphology. It has not been fully understood whether the grafted neural stem cells could differentiate into the site-specific neurons. This experiment is to investigate the neuronal differentiation of the neural stem cells derived from a human fetal brain after transplanted into young rats' brains, to study the possibility of cell-replacement therapy for children's brain disorders with neural stem cells. METHODS: Experiments were performed at the Cell Laboratory of Naval General Hospital from April to July 2007. ①Human fetal brain tissues of 16 week gestation were provided by Department of Gynaecology and Obstetrics of Naval Hospital. Pregnant woman and family members signed an informed consent. Experimental intervention was approved by Hospital Ethical Committee. Fourteen clean brood young SD rats aged 10 days, irrespective of gender, were provided by Experimental Animal Center of Medical College of Peking University. Animal intervention met the animal ethical standards. ②The neural stem cell spheres were derived from the fetal brain tissues of 16 week gestation. The differentiation multipotency of the neurosphere was identified when cultured in a child's cerebrospinal fluid (CSF). The neurospheres cultured in vitro for 14 days were injected into the lateral ventricles of young rats of 10 days old. The rats were respectively killed at days 4, 7 and 14 after transplantation. The special immuno-fluorescent assays were performed using anti-human neurofilament (anti-hNF) to show the location and morphology of graft neurons. RESULTS: ①The typical floating neurospheres were obtained, with the potency to differentiate into neurons, astrocytes and oligodendrocytes. ②The neuronal differentiation of grafts was detected with the mixture of three monoclonal antibodies against human neurofilament. Four days after transplantation, the immune response positive cells lied within the granule cell layer of cerebral cortex were shown in the shape of granule cells, or within the pyramid cell layer in the shape of pyramid cells with long processes, and the interneuron-like cells also were seen. The Purkinje cells arranging in a monolayer were detected in the cerebellum. Compared the results at different time points, the location of grafts were the same. The graft cells were less and the processes were longer over time. CONCLUSION: The in vitro cultured neurosphere cells can migrate into brain tissues and differentiate into site-specific neurons in shape after transplanting into the lateral ventricles of young rats. It is suggested that the host brain tissue microenvironment played an important role in guiding the graft differentiation into neurons. The results have an important significance for understanding cell replacement of developing brain disorders.