On day −1, 400,000 KOLF2-1J or SFC065 (kindly provided by the laboratory of C. Klein) hiPSC/cm2 were seeded in Matrigel-coated 6-well plate wells in StemFlex medium supplemented with 10 μM RI. On day 0, medium was switched to Neurobasal containing 0.5x B27 supplement without vitamin A, 0.5x N2, GlutaMAX, Pen/strep, non-essential amino acids, and LDN193189 (500 nM, Sigma), SB431542 (10 μM, Tocris), SHH-C24II (200 ng/ml, Miltenyi Biotec), Purmorphamine (0.7 μM, Sigma) and 0.7 μM CHIR99021 (Stemcell Technologies). CHIR99021 concentration was raised to 3 μM from day 4 to day 11, moment at which it was withdrawn from the medium. LDN193189 (500 nM, Sigma), SB431542 (10 μM, Tocris), SHH-C24II (200 ng/ml, Miltenyi Biotec), Purmorphamine (0.7 μM, Sigma) were withdrawn from the medium at day 7 and FGF8b (100 ng/mL, R&D Systems) was introduced from day 9 until day 16. At day 11, medium was shifted to Neurobasal-A medium (Life Technologies; 10888-022) supplemented with 1x B27 without Vit. A (Life Technologies; 12587010), 1x GlutaMAX (Life Technologies; 35050-038), 1x PenStrep (Life Technologies; 15140-122), 10 ng/mL BDNF (R&D Systems; 248-BDB-050/CF), 10 ng/mL GDNF (R&D Systems; 212-GD-010), 200 μM ascorbic acid, 0.5 mM dbcAMP (Sigma-Aldrich), 10 μM DAPT (Tocris; 2634). On day 17, ventral midbrain neural progenitors were cryopreserved.
Pantazis C.B., Yang A., Lara E., McDonough J.A., Blauwendraat C., Peng L., Oguro H., Kanaujiya J., Zou J., Sebesta D., Pratt G., Cross E., Blockwick J., Buxton P., Kinner-Bibeau L., Medura C., Tompkins C., Hughes S., Santiana M., Faghri F., Nalls M.A., Vitale D., Ballard S., Qi Y.A., Ramos D.M., Anderson K.M., Stadler J., Narayan P., Papademetriou J., Reilly L., Nelson M.P., Aggarwal S., Rosen L.U., Kirwan P., Pisupati V., Coon S.L., Scholz S.W., Priebe T., Öttl M., Dong J., Meijer M., Janssen L.J., Lourenco V.S., van der Kant R., Crusius D., Paquet D., Raulin A.C., Bu G., Held A., Wainger B.J., Gabriele R.M., Casey J.M., Wray S., Abu-Bonsrah D., Parish C.L., Beccari M.S., Cleveland D.W., Li E., Rose I.V., Kampmann M., Aristoy C.C., Verstreken P., Heinrich L., Chen M.Y., Schüle B., Dou D., Holzbaur E.L., Zanellati M.C., Basundra R., Deshmukh M., Cohen S., Khanna R., Raman M., Nevin Z.S., Matia M., Van Lent J., Timmerman V., Conklin B.R., Chase K.J., Zhang K., Funes S., Bosco D.A., Erlebach L., Welzer M., Kronenberg-Versteeg D., Lyu G., Arenas E., Coccia E., Sarrafha L., Ahfeldt T., Marioni J.C., Skarnes W.C., Cookson M.R., Ward M.E, & Merkle F.T. (2022). A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell stem cell, 29(12), 1685-1702.e22.
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