EFFECTS OF T_3 ON DIFFERENTIATION OF HUMAN NEURAL STEM CELLS TO OLIGODENDROCYTE / 解剖学报

ABSTRACT

Objective The aim of the study was to assess the human neural stem cells (NSCs) induced by mitogens in vitro and investigate effects of TH(T 3) on differentiation. Methods The cerebral hemispheres of human fetal at 10-12 weeks of gestation were minced and mechanically dissociated. Cells were seeded at a concentration of 10 5 per ml into defined medium that consisted of DMEM/F12 Epidermal growth factor (EGF,20??g/L) and basic fibroblast growth factor (bFGF,20??g/L) were added respectively as mitogens for 7 days. In some cases, triiodothyronine (T 3, 30??g/L) was added through the culture period. Upon differentiation, the cell cultures were exposured to a substrate and treated with or without T 3 after removing the mitogens. To detect the phenotypes of differentiated cells, immunochemistry staining was performed with the antibody to NF 200, GFAP, MBP and Gal C. To recognize the different stages of oligodendrocytes development, the antibodies O4 and A2B5 were used. The proportion of each neural cell type was determined by counting positive cells in standardized fields at 40? magnifications. Results NSCs induced by EGF and/or bFGF grew in culture as free floating spheres(neurospheres) and were immunoreactive for the intermediate filament nestin. After removing EGF and bFGF, the cells cease mitosis and can be induced to differentiate into neurons, astrocytes, and oligodendrocytes regardless of whether T 3 was presented. T 3 favored an neuroglia cell fate especially when combining EGF with T 3 and differentiated cells appeared more early when added of T 3.MBP positive cell is over 80%. The O4 and A2B5 positive cells can be observed at early stage of differentiation only after process grew from neurospheres after adhesion to certain substrate, which indicated that the neuronal network could promote the maturity of oligodendrocyte.Discussion The timing of NSCs differentiation depends on both intracellular mechanisms and extracellular signals. TH is such a signal to activate the effector component of a “clock” mechanism that induces oligodendrocyte differentiation after a limited number of cell divisions.[