Neural Stem Cell Differentiation from iPSCs

Human NSCs were derived by differentiating human iPSC HEL24.3^{45 (link)}, and HEL46.11 lines using small molecule cocktail as described elsewhere^{46 (link)}, with minor adjustments. Briefly, iPSCs were detached with StemPro Accutase (Thermo Fisher Scientific) and dissociated gently into single cells suspension in hES-medium in the presence of 5 μM ROCK inhibitor (ROCKi; Y-27632, Selleckchem), 10 μM SB431542 (SB; S1067, Selleckchem), 1 μM dorsomorphin (DM; P5499-5MG, Sigma), 3 μM CHIR-99021 (CHIR; Tocris) and 0,5 μM purmorphamine (PMA; 04-0009, Stemgent) After 2 days, medium was changed to N2B27 medium (DMEM/F12:Neurobasal (1:1) supplemented with N2 and B27 without vitamin A, NEAA, PenStrep (all Thermo Fisher Scientific) and heparin (2 µg/ml; H3149-50KU, Sigma)) containing the same small molecule cocktail as above. On day 4, SB and DM were withdrawn and 150 µM ascorbic acid (AA) was added to N2B27. On day 6, the neurospheres were dissociated with 1 ml pipette and plated on Matrigel in N2B27 media containing AA, CHIR and PMA (growth media). First two passages were split at 1:3 ratio and cells were plated into growth media containing 5 μM ROCKi, which was removed next day. Later passages were split with 1:10 and 1:20 ratio using StemPro Accutase. Media were changed every other day.

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Weltner J., Balboa D., Katayama S., Bespalov M., Krjutškov K., Jouhilahti E.M., Trokovic R., Kere J, & Otonkoski T. (2018). Human pluripotent reprogramming with CRISPR activators. Nature Communications, 9, 2643.

Publication 2018

StemPro Accutase Y-27632 S1067 CHIR-99021 (CHIR; PenStrep H3149-50KU

Accutase Ascorbic acid Cells Chir 99021 Dorsomorphin Growth media Heparin Human Ipscs Matrigel Purmorphamine Sb431542 Vitamin a Y 27632

Corresponding Organization : Helsinki University Hospital

Other organizations : Karolinska Institutet

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Variable analysis

independent variables

Small molecule cocktail (5 μM ROCK inhibitor (ROCKi), 10 μM SB431542 (SB), 1 μM dorsomorphin (DM), 3 μM CHIR-99021 (CHIR), and 0.5 μM purmorphamine (PMA))
Withdrawal of SB and DM on day 4
Addition of 150 μM ascorbic acid (AA) on day 4
CHIR and PMA in growth media

dependent variables

Differentiation of human iPSC HEL24.3 and HEL46.11 lines into human neural stem cells (NSCs)

control variables

Dissociation of iPSCs into single cells
Plating of neurospheres on Matrigel
Media changes every other day
Passage ratios (1:3 for first two passages, 1:10 and 1:20 for later passages)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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