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.
Macs ips brew media
Manufactured by Miltenyi Biotec
MACS iPS-Brew media is a serum-free and animal component-free culture medium designed for the growth and maintenance of human induced pluripotent stem (iPS) cells. It supports the undifferentiated expansion and self-renewal of iPS cells.
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3 protocols using macs ips brew media
1
Differentiation of iPSCs to Spinal Motor Neurons
2
Differentiation of iPSCs to Motor Neurons
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Human induced pluripotent stem cell lines were routinely cultured on Matrigel-coated dishes in MACS iPS-Brew media (Miltenyi Biotec) and passaged using ReLESR (Stem Cell Technologies) on a weekly basis. To induce motor neuron differentiation, pluripotent colonies were detached from the culture dish using Accutase and exposed to neural induction media consisting of 4.25 μM CHIR99021 and 0.5 μM LDN-193189 for the first 10 days for culture on Matrigel-coated dishes. 1 μM Retinoic acid (RA) was supplemented into this media from days 3 to 10. From days 10 to 17, cells were cultured in motor neuron patterning media consisting of 1 μM RA and 1 μM Purmorphamine. Subsequently, from days 18 to 28, the adherent culture was dissociated into single cells and re-plated onto Matrigel-coated dishes in media consisting of 10 ng/ml BDNF and 10 ng/ml GDNF. N2B27 media: 50% DMEM/F12, 50% Neurobasal medium, 1X N2 supplement, 1X B27 supplement, 1X NEAA and 1X Glutamax.
3
Spinal organoid derivation from iPSCs
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Wild-type BJ fibroblast-derived iPSCs (BJ-iPS) and SMA patient-derived iPSCs (Type II SMA 1-51 N and Type I SMA 1-38 G) were cultured feeder-free on Matrigel-coated plates in MACS iPS-Brew media (Miltenyi Biotec). Routine passaging using ReLeSR (Stem Cell Technologies) is performed once every 6-7 days. Pluripotent stem cells were differentiated towards the spinal motor neuron fate following established protocols described previously1 (link). Spinal organoids were made by dissociating iPS cells into single cells, and seeded either 10,000 or 30,000 cells per well in a 96-well low-attachment plate. Eventually we used 30,000 cells per well because that resulted in better derivation of mature spinal cell types (Supplementary Figure S1 ). The embryoid bodies were then encapsulated in 15 μl Matrigel droplets at day 10 before transferring to spinner flasks at day 14 for neuronal maturation in the presence of growth factors BDNF and GDNF. Organoids can be maintained for at least 90 days, although in this manuscript, organoids were harvested by day 42 for analysis (Supplementary Figure S1 ).
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