Wild-type BJ fibroblast-derived iPSCs (BJ-iPS) were cultured feeder-free on matrigel-coated plates in MACS iPS-Brew media (Miltenyi Biotec). Routine passaging using ReLeSR (Stem Cell Technologies) was performed once every 6-7 days. Pluripotent stem cells were differentiated towards the spinal motor neuron fate following established protocols described previously. Briefly, we first neutralized the BJ-iPS by activating Wnt pathways with CHIR99021 treatment (4.25 μM, Miltenyi Biotec) while blocking Bone Morphogenic Protein (BMP) signaling by LDN-193189 treatment (0.5 μM, Miltenyi Biotec) at the same time. At day 3, variable concentrations of retinoic acid and GDF11 were added to initiate the rostral-caudal patterning, in the presence of fixed concentration of Purmorphamine (1 μM, Miltenyi Biotec), a Sonic Hedgehog pathway agonist, as a ventralizing signal. Neurotrophic factors, BDNF (20 ng/ml, Miltenyi Biotec) and GDNF (20 ng/ml, Miltenyi Biotec), were added to the neuronal cultures at day 17 to promote neuronal maturation into motor neurons.
Purmorphamine
Manufactured by Miltenyi Biotec
Purmorphamine is a small molecule compound that functions as a hedgehog pathway agonist. It is commonly used in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Miltenyi Biotec (2 mentions)
Relesr, by STEMCELL (1 mentions)
Macs ips brew media, by Miltenyi Biotec (1 mentions)
Brain derived neurotrophic factor (bdnf), by Miltenyi Biotec (1 mentions)
Ldn 193189, by Miltenyi Biotec (1 mentions)
Laminin, by Merck Group (1 mentions)
N2 supplement, by Merck Group (1 mentions)
Pen strep, by Merck Group (1 mentions)
Glutamax, by Merck Group (1 mentions)
Dmem f12 neurobasal, by Merck Group (1 mentions)
B27 without vitamin a, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12 neurobasal, by Thermo Fisher Scientific (1 mentions)
2 protocols using purmorphamine
1
Differentiation of iPSCs to Spinal Motor Neurons
2
Generation and Differentiation of smNPCs
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Generation of smNPCs from iPSCs was done according to a previously published protocol (68 ). smNPCs were cultured in Matrigel-coated 6-well plates in medium composed of DMEM-F12/neurobasal (50:50), 1% N2 supplement, 2% B27 without vitamin A (all Gibco), 0.5 μM purmorphamine, 3 μM CHIR99021 (both from Miltenyi Biotec), and 64 μg/ml LAAP (Sigma). siRNA transfection of smNPCs was performed as described for HeLa cells. To induce smNPC differentiation, medium was changed to differentiation medium containing DMEM-F12/neurobasal, 1% N2 supplement, 2% B27, 2 mM Glutamax, 1% Pen/Strep, and 1 μg/ml laminin (Sigma). Cells were differentiated under these conditions for 25 to 30 days before being collected for co-IP experiments.
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