NMP Differentiation from Embryonic Stem Cells

NMP differentiation was based on the published protocols^{20 (link),56 (link)} with minor modifications. In preparation for differentiation, ESCs were feeder-depleted and plated in gelatin-coated (0.1% Sigma, G1890-100G) 6-well plates (Falcon, 353046) at a density of 8×10³ cells/cm² in ES medium. On D0 of differentiation media was changed to N2B27 media (Composition: 49.5% Advanced Dulbecco’s Modified Medium F-12 (Gibco, 12634028); 49% Neurobasal medium (Gibco, 21103049); 0.5% N2-supplement (Gibco, 17502001); 1% B27-supplement (Gibco, 17504044)) supplied with 1x Glutamax (Gibco, 17504044), 40 µg/ml BSA Fraction V (Gibco, 15260037) and 100 mM 2-Mercaptoethanol (Gibco, 21985-023)) supplemented with 10 ng/ml hFGF-2 (Miltenyi Biotec, 130-104-925). The media was further supplemented with 5 µM CHIR99021 (StemMACS, 130-103-926) on D2 and with or without 50 ng/ml hGdf11 (130-105-776, Miltenyi Biotec) on D3. Throughout differentiation, the medium was refreshed every 24 h. NMP identity at D3 was routinely confirmed by co-expression analysis of Sox2 and T/Bra using immunofluorescence.

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Chang Y.C., Manent J., Schroeder J., Wong S.F., Hauswirth G.M., Shylo N.A., Moore E.L., Achilleos A., Garside V., Polo J.M., Trainor P, & McGlinn E. (2022). Nr6a1 controls Hox expression dynamics and is a master regulator of vertebrate trunk development. Nature Communications, 13, 7766.

Publication 2022

Neurobasal medium N2-supplement B27-supplement Glutamax BSA Fraction V 2-Mercaptoethanol hFGF-2 hGdf11

Chir99021 Escs Gelatin Immunofluorescence Mercaptoethanol Sox2

Corresponding Organization :

Other organizations : EMBL Australia, Monash University, Australian Regenerative Medicine Institute, Stowers Institute for Medical Research

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Variable analysis

independent variables

Presence or absence of 50 ng/ml hGdf11 on D3 of differentiation

dependent variables

NMP identity at D3 (confirmed by co-expression analysis of Sox2 and T/Bra using immunofluorescence)

control variables

ESCs were feeder-depleted and plated in gelatin-coated (0.1% Sigma, G1890-100G) 6-well plates at a density of 8×10^3 cells/cm^2 in ES medium
On D0 of differentiation, media was changed to N2B27 media supplemented with 1x Glutamax, 40 µg/ml BSA Fraction V, and 100 mM 2-Mercaptoethanol
N2B27 media was further supplemented with 10 ng/ml hFGF-2 on D0 and 5 µM CHIR99021 on D2
Throughout differentiation, the medium was refreshed every 24 h

controls

Positive control: NMP identity at D3 was routinely confirmed by co-expression analysis of Sox2 and T/Bra using immunofluorescence

Annotations

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