In Vitro Neural Cell Differentiation Derived from Human Embryonic Stem Cells: Effects of PDGF-bb and BDNF on the Generation of Functional Neurons / 대한불임학회지

ABSTRACT

OBJECTIVE: This study was to investigate the generation of the functional neuron derived from human embryonic stem(hES,MB03) cells on in vitro neural cell differentiation system. METHODS: For neural progenitor cell formation derived from hES cells, we produced embryoid bodies (EB for 5 days, without mitogen) from hES cells and then neurospheres (for 7~10 days, 20 ng/ml of bFGF added N2 medium) from EB. And then finally for the differentiation into mature neuron, neural progenitor cells were cultured in i) N2 medium only (without bFGF),ii) N2 supplemented with 20 ng/ml platelet derived growth factor-bb (PDGF-bb) or iii) N2 supplemented with 5 ng/ml brain derived neurotrophic factor (BDNF) for 2 weeks. Identification of neural cell differentiation was carried out by immunocytochemistry using betaIII-tubulin (1100) and GFAP (1500). Also, generation of functional neuron was identified using anti-glutamate (Sigma, 11000), anti-GABA (Sigma, 11000), anti-serotonin (Sigma, 11000) and anti-tyrosine hydroxylase (Sigma, 11000). RESULTS: In vitro neural cell differentiation, neurotrophic factors (PDGF and BDNF) treated cell groups were high expressed MAP-2 and GFAP than non-treated cell group. The highest expression pattern of MAP-2 and betaIII-tubulin was indicated in BDNF treated group. Also, in the presence of PDGF-bb or BDNF, mostof the neural cells derived from hES cells were differentiated into gultamate and GABA neuron in vitro. Furthermore, we confirmed that there were a few serotonin and tyrosine hydroxylase positive neuron in the same culture environment. CONCLUSION: This results suggested that the generation of functional neuron derived from hES cells was increased by addition of neurotrophic factors such as PDGF-bb or BDNF in b-FGF induced neural cell differentiation system and especially gultamate and GABA neurons were mainly produced in the system.