Derivation of Neural Stem Cells from hESCs

hESCs were digested into small clumps for suspension culture on ultra-low attachment plates (Corning, 3471) in the NESC-derived medium [26 (link)], which is composed of Advance DMEM/F12 (Gibco, 10565-018): Neurobasal media (Gibco, 21103-049) (1:1) supplemented with 1% N2 (Gibco, 17502-048), 2% B27 (Gibco, 17504-044), 1% Glutmax (Gibco, 35050-061), 10 ng/mL OsrbFGF (Oryza sativa recombinant human basic fibroblast growth factor, Wuhan Healthgen, China, HYC005M01), 3 μM CHIR99021 (Selleck, S2924), 5 μM SB431542 (Cellagen technology, C7243), 0.2 μM Compound E (Calbiochem, 565790), 0.1 μM LDN193189 (Selleck, S2618), and 0.1 mM β-mercaptoethanol (Sigma, M3148). After suspension culture for 6 days, neuron bodies (NBs) were digested into single cells and inoculated into ultra-low attachment plates with CHbFSB+LIF culture medium. The CHbFSB+LIF culture medium [26 (link), 28 (link), 31 (link)] is composed of Neurobasal medium, 1% N2, 2% B27, 1% NEAA (Gibco, 11140-050), 1% Glutmax, 3 μM CHIR99021, 5 μM SB431542, and 10 ng/ml OsrbFGF surplus with 1000 U/ml hLIF (Millipore, LIF1050).

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Zhao S., Duan K., Ai Z., Niu B., Chen Y., Kong R, & Li T. (2020). Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells. Stem Cell Research & Therapy, 11, 431.

Publication 2020

ultra-low attachment plates Advance DMEM/F12 Neurobasal media Glutmax CHIR99021 SB431542 LDN193189 β-mercaptoethanol NEAA LIF1050

Bodies Cells Chir99021 Compound e Hescs Ldn193189 Mercaptoethanol Neuron Oryza sativa Recombinant human basic fibroblast growth factor Sb431542

Corresponding Organization :

Other organizations : Kunming University of Science and Technology

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Variable analysis

independent variables

Culture medium composition
Culture duration

dependent variables

Neuron body (NB) formation

control variables

Use of ultra-low attachment plates
Cell type (hESCs)
Presence of supplements in the media (N2, B27, Glutmax, β-mercaptoethanol)
Concentrations of growth factors and small molecules (OsrbFGF, CHIR99021, SB431542, Compound E, LDN193189)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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