Effects And Mechanisms Of Valproic Acid On Differentiation And Apoptosis Of Human Hippocampal Neural Progenitor Cells

Valproic acid (VPA) is one of the major commonly used antiepileptic drugs (AEDs) at present, but clinical researches suggested that it had potential neurotoxicity to developing brains, and that children exposed to VPA in utero had a high rate for neural tube defects, developmental delays, mental retardation, cognitive impairment and other behavioral deficits. Animal researches also found that VPA may have side effects on several important processes of brain development, such as cell proliferation, migration, differentiation and apoptosis et al. However, clinical researches didn't or couldn't exclude the interference factors as seizures, family environment, potential developmental deficits et al, and animal researches have species variations and can't reflect the actual conditions in human body, on the other hand, we aren't able to explore the detailed mechanisms on a molecular level and to reach a comprehensive conclusion through these studies.So in this study we separated and cultured neural progenitor cells from human fetal hippocampus in vitro, and explored the effect of VPA of clinical relevant concentrations on the differentiation and apoptosis of progenitor cells and the role of astrocytes played in VPA-induced neural cell apoptosis.PartⅠThe separation, cultivation and differentiation of neural progenitor cells from the hippocampus of human fetusesObjective: To explore the cultivation and committed differentiation of neural progenitor cells from the hippocampus of human fetuses, and to establish an appropriate cellular model for in vitro experiments. Methods: Aborted human fetuses aged from 10 to 14 weeks post-conception were sterilized with 75% alcohol, and hippocampus tissues were dissected and mechanically dissociated by repetitive blowing with a 200 ul pipette into single cell suspension. Primary cells were cultured at 1×106 cells/ml in DMEM/F12 media (Gibco, USA) supplemented with L-glutamine (1%, Gibco, USA), N-2 ( 1%, Gibco, USA), B27 ( 2%,Gibco, USA), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) ( 20ng/ml, Pepro Tech Inc, USA); To select for progenitor cells more committed to a neuronal linage, bFGF was added to expand some of the primary cells. After the third passage in culture, the neurospheres were seeded to coverslips in 24-well plates and exposed to a differentiation media (DMEM/F12 containing 1% L-glutamine, 1% N-2, 2% B27, and 1ng/ml bFGF). Results: After exposure of the neurosphere cultures from the third passage to differentiation media, cells began to migrate out of neurospheres 48 h after induction. A number of the migrating cells gradually displayed distinct neuronal morphology with round or elliptic somas from which two or three long neurites were radiated. A second population of cells displayed an irregular astrocytic-like appearance with large and flat somas from which processes were radiated. After 3-days of differentiation, immunofluorescent microscopy showed that the former were Tuj1 positive neurons and the latter were GFAP positive astrocytes. Progenitor cells amplified with EGF+bFGF could differentiate into both neurons and astrocytes, and the ratio of them was 38.8%±4.2% and 57.3%±2.9% respectively, while neural progenitor cells amplified with bFGF mostly differentiated into neurons(81.2%±3.9%), few astrocytes were detected. But neuronal differentiation and development was impaired in the absence of astrocytes, less neurons migrated out of the neurospheres, and neurite outgrowth was apparently shorter. Conclusion: 1. Through the above experiment, we successfully obtained neural progenitor cells with the ability of self-renewing and multiple differentiation; 2. EGF+bFGF generated neural progenitor cells differentiated into both neurons and astrocytes, while bFGF generated progenitor cells mostly differentiated into neurons. The cellular model provides experimental platform for further studies. PartⅡEffects and mechanisms of valproic acid on the differentiation and apoptosis of hippocampal neural progenitor cellsObjective: To study the effect of valproic acid (VPA) on the differentiation and apoptosis of neural progenitor cells and explore the mechanism of VPA induced neural cell apoptosis. Methods: The neurospheres were seeded to coverslips in 24-well plates and exposed to a differentiation media (DMEM/F12 containing 1% L-glutamine, 1% N-2, 2% B27, and 1ng/ml bFGF) and VPA at either 0, 250μM, 500μM or 1mM for 7 days. To confirm the effect of TNF-αon neuronal apoptosis, 2.5μg/ml purified mouse anti-human TNF-αantibody, or 50pg/ml and 100pg/ml of TNF-αwere added to some cultures to block or to simulate the effect of TNF-α. Differentiating neurons and astrocytes were identified by detection of the expression of Tuj1 and GFAP by immunofluorescence. Apoptosis was determined using the Hoechst33342 labeling protocol. Activated caspase-3 was detected by immunofluorescence and the level of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in the culture supernatant was detected with ELISA detection kits. Results: 1. VPA significantly accelerated the differentiation process, a mass of cells migrated out of the neurospheres upon VPA exposure 24h after induction of differentiation, 3 days later more cells migrated out, and the cell processes grew longer upon exposure, the effect of 500μM and 1mM VPA was more prominent than 250μM; After 3 days of differentiation, immunofluorescence showed that on EGF+bFGF generated neural progenitor cells, VPA could apparently promoted neuronal differentiation and inhibited astrocyte differentiation, 500μM VPA had the most significant effect, upon which the percentage of neurons increased to 59.2%±2.1% (vs 38.8%±4.2% in the control cultures) and astrocytes decreased to 36.4%±3.4% (vs 57.3%±2.9% in the control). Moreover, the effect displayed a concentration and time dependent manner, higher concentration and longer exposure time caused more apparent effect; In bFGF generated neural progenitor cells, VPA could apparently promote neuronal differentiation and development, more neurons migrated out of the neurospheres, and neurites grew longer. 2. 3 days later, with exposure time prolonging, on EGF+bFGF generated NPCs, differentiating cells gradually exhibited on bFGF generated NPCs, VPA exposure did not cause increased apoptosis, only at 1 mM and 7 days exposure, a slight increase in the apoptotic ratio (15.0%±1.6% vs 6.0%±2.0% in the control) was observed; 3. Immunofluorescent microscopy showed that most cells with apoptotic morphologic nuclei were Tuj1-positive neurons, that also co-expressed cleaved caspase-3(Asp175); GFAP-positive astrocytes did not show any apoptosis changes, but it did show morphological changes, and they acquired much longer processes via the shrinkage of the cytoplasm, compared with the irregular and flat appearance in control cells, which suggesting activation. 4. Detection of TNF-αand IL-1βwith an ELISA detection method showed that exposure of the differentiating EGF+bFGF generated NPCs to VPA produced significant amount of TNF-αbut no IL-1β, while none of them was detected in the similarly exposed bFGF generated NPCs. Moreover, TNF-αlevel correlated with the percentage of apoptotic neurons. 5. TNF-αantibody partially prevented DNA-fragmentation, as apoptosis decreased to 15.3%±1.9% at day 5 and 20.3%±2.4% at day 7 respectively, compared with that of 25.6%±0.2% and 37.4%±1.7% in the control cells; TNF-αat concentrations of 50pg/ml and 100pg/ml induced apoptosis in 16.0%±3.0% and 30.0%±2.4% of the neuronal cells respectively. Conclusion: 1. VPA could apparently accelerate the differentiation of neural progenitor cells, interfere with the orientation of differentiation, it significantly promoted neuronal differentiation and inhibited astrocyte differentiation; 2. VPA selectively induced neuronal apoptosis, which was related with astrocyte activation and the release of TNF-α; 3. These effects displayed an apparent dose and time dependent manner, 500μM and 1mM VPA exhibited a more apparent effect than 250μM.