Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells
Figure 1
Neurosphere formation of marmoset ESCs.
(A) Protocol to derive neurospheres from marmoset ESCs by EB formation. EBs were cultured in suspension in ultra-low cluster dishes for 2 weeks in the presence of 3 µM dorsomorphin or 1×10−6 M RA. Dorsomorphin or RA were added on day 1 or 5 of EB formation, respectively. EBs were then dissociated and cultured in suspension for 12–14 days to form neurospheres in MHM medium containing 2% B27 and 20 ng/ml FGF-2. Primary neurospheres were dissociated and cultured in suspension again with FGF-2 to form secondary neurospheres. (B) Neurosphere formation rates are presented as the percentages of neurospheres among total cells plated. EBs treated with 3 µM dorsomorphin or 1×10−6 M RA were dissociated and cultured in MHM medium containing 2% B27 and 20 ng/ml FGF-2 at a density of 2.5×104 cells/ml in an ultra-low cluster 96-well plate for 1 week, and then neurospheres larger than 50 µm in diameter were counted. Data are presented as the means ± SEM (n = 3). (C) Representative morphologies of EBs, primary neurospheres and secondary neurospheres under each condition. Scale bars, 100 µm for EBs, 200 µm for neurospheres.