Pluripotent cells were differentiated in neural stem cells (NSCs), using a modified dual SMAD inhibition protocol [20 (link)]. In short, 18,000 cells/cm2 were plated on Matrigel-coated cell culture dishes in mTeSR-1 medium in the presence of 10 µM Y27632. When cells reached 90% confluency, the medium was switched to differentiation medium (KnockOut DMEM (Gibco), 15% KnockOut serum replacement (Gibco), 2 mM l-glutamine (Gibco), MEM non-essential amino acids (Sigma), 0.1 mM β-mercaptoethanol, 100U/ml penicillin and 100 µg/ml streptomycin) supplemented with 2 µM A 83-01 (Tocris) and 2 µM Dorsomorphin (Sigma-Aldrich). At day 6, medium was changed to an equal ratio of differentiation medium and NSC medium (KnockOut DMEM-F12 (Gibco), 2 mM l-glutamine (Gibco), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Peprotech), 2% StemPro Neural supplement (Gibco), 100U/ml penicillin and 100 µg/ml streptomycin) supplemented with 2 µM A 83-01 (Tocris) and 2 µM Dorsomorphin (Sigma-Aldrich). At day 10, cells were passaged (NSC p = 0) using Accutase (Sigma) and maintained in NSC medium. We used commercially available H9-derived NSCs (Gibco) as a control (a kind gift from Raymond Poot, Rotterdam).
Perenthaler E., Nikoncuk A., Yousefi S., Berdowski W.M., Alsagob M., Capo I., van der Linde H.C., van den Berg P., Jacobs E.H., Putar D., Ghazvini M., Aronica E., van IJcken W.F., de Valk W.G., Medici-van den Herik E., van Slegtenhorst M., Brick L., Kozenko M., Kohler J.N., Bernstein J.A., Monaghan K.G., Begtrup A., Torene R., Al Futaisi A., Al Murshedi F., Mani R., Al Azri F., Kamsteeg E.J., Mojarrad M., Eslahi A., Khazaei Z., Darmiyan F.M., Doosti M., Karimiani E.G., Vandrovcova J., Zafar F., Rana N., Kandaswamy K.K., Hertecant J., Bauer P., AlMuhaizea M.A., Salih M.A., Aldosary M., Almass R., Al-Quait L., Qubbaj W., Coskun S., Alahmadi K.O., Hamad M.H., Alwadaee S., Awartani K., Dababo A.M., Almohanna F., Colak D., Dehghani M., Mehrjardi M.Y., Gunel M., Ercan-Sencicek A.G., Passi G.R., Cheema H.A., Efthymiou S., Houlden H., Bertoli-Avella A.M., Brooks A.S., Retterer K., Maroofian R., Kaya N., van Ham T.J, & Barakat T.S. (2019). Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases. Acta Neuropathologica, 139(3), 415-442.
Other organizations :
Erasmus MC, King Faisal Specialist Hospital & Research Centre, University of Novi Sad, Amsterdam Neuroscience, Amsterdam University Medical Centers, University of Amsterdam, McMaster Children's Hospital, Stanford University, Sultan Qaboos University, Sultan Qaboos University Hospital, Radboud University Nijmegen, Radboud University Medical Center, Mashhad University of Medical Sciences, University of Sistan and Baluchestan, St George's, University of London, National Hospital for Neurology and Neurosurgery, University College London, Centogene (Germany), Tawam Hospital, King Saud University, Shahid Sadoughi University of Medical Sciences and Health Services, Yale University, Choithram Hospital and Research Centre
Differentiation of pluripotent cells into neural stem cells (NSCs)
control variables
Plating density: 18,000 cells/cm^2
Cell culture surface: Matrigel-coated
Cell culture medium: mTeSR-1 with 10 µM Y27632
Differentiation medium: KnockOut DMEM, 15% KnockOut serum replacement, 2 mM L-glutamine, MEM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100U/ml penicillin and 100 µg/ml streptomycin, supplemented with 2 µM A 83-01 and 2 µM Dorsomorphin
NSC medium: KnockOut DMEM-F12, 2 mM L-glutamine, 20 ng/ml bFGF, 20 ng/ml EGF, 2% StemPro Neural supplement, 100U/ml penicillin and 100 µg/ml streptomycin, supplemented with 2 µM A 83-01 and 2 µM Dorsomorphin
Commercially available H9-derived NSCs (Gibco) as a control
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