The system for directional and stable differentiation of amniotic fluid stem cells into neuron-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers. Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation, a high risk of tumor development in vivo limits the further study. OBJECTIVE: To establish a stable system for sorting, culture and neuronal differentiation of amniotic fluid stem cells, and to explore the feasibility as seed cells for neuronal regeneration. METHODS: Amniotic fluid sample (10 mL) was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance, and amniotic fluid stem cells were isolated by c-Kit magnetic beads. The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence. The expression levels of c-Kit, Oct-4, Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR. Then, the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures. Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method. The expression levels of Neuro D and Tuj1 were observed by immunofluorescence. RESULTS AND CONCLUSION: (1) About 1% of amniotic fluid stem cells were positive for c-Kit. (2) (75.0±4.6)% of amniotic fluid stem cells expressed Oct-4 and (86.0±2.8)% of the cells expressed Sox2. (3) The expression levels of c-Kit, Oct-4, Sox2 and Nestin detected by RT-PCR did not change with passage times. (4) Embryoid bodies-like structures formed after hanging drop culture. (5) Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1, but without the typical morphological features. RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages. (6) Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin factor 3 in two stages, and could express neural stem cell marker Neuro D and neuronal marker Tuj1.