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Spontaneous and lineage-directed differentiation of hESCs and hiPSCs

JJ Jiwon Jang
KK Kenneth S. Kosik

Last updated date: Jul 3, 2021 Views: 810 Forks: 0

original An abbreviated version of this protocol was published in PLoS Biology
Control over single-cell distribution of G1 lengths by WNT governs pluripotency
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Spontaneous differentiation

1. Human PSCs (hPSCs) including hESCs and hiPSCs are cultured in mTeSR1 (Stem cell technologies, 85850) or Essential 8 (Thermo Fisher, A1517001) medium on Matrigel (Corning, 354277)-coated 6-well plates.

2. When the culture reaches 50% confluency, the cells are washed with 1ml PBS and differentiated with 4 ml of the KOSR medium.

3. The cells are differentiated for 8 days with the fresh medium every other day. 

 

KOSR medium

ReagentsVolumeProduct information
DMEM/F-12, GlutaMAX™ supplement41.5mlThermo Fisher, 10565018
KnockOut™ Serum Replacement7.5mlThermo Fisher, 10828010
MEM Non-Essential Amino Acids Solution (100X)0.5mlThermo Fisher, 11140050
2-Mercaptoethanol100ulThermo Fisher, 21985023
Penicillin-Streptomycin (10,000 U/mL)0.5mlThermo Fisher, 15140122

 

Neuroectoderm-directed differentiation

1. Human PSCs (hPSCs) including hESCs and hiPSCs are cultured in mTeSR1 or Essential 8 medium on Matrigel-coated 6-well plates. 

2. When the culture reaches 70~80% confluency, the cells are washed with 1ml PBS and differentiated with 2ml of the SMADi medium. 

3. The cells are differentiated for 5 days with the fresh medium everyday.

 

SMADi medium

ReagentsVolumeProduct information
DMEM/F-12, GlutaMAX™ supplement41.5mlThermo Fisher, 10565018
KnockOut™ Serum Replacement7.5mlThermo Fisher, 10828010
MEM Non-Essential Amino Acids Solution (100X)0.5mlThermo Fisher, 11140050
2-Mercaptoethanol100ulThermo Fisher, 21985023
Penicillin-Streptomycin (10,000 U/mL)0.5mlThermo Fisher, 15140122
Recombinant Human Noggin200 ng/mlPeprotech, 120-10C
SB 43154210μMTocris, 1614

 

Mesendoderm-directed differentiation

1. Human PSCs (hPSCs) including hESCs and hiPSCs are cultured in mTeSR1 or Essential 8 medium on Matrigel-coated 6-well plates. 

2. When the culture reaches 10~20% confluency, the cells are washed with 1ml PBS and differentiated with 2ml of the BMP4 medium. 

3. The cells are differentiated for 5 days with the fresh medium everyday.

 

BMP4 medium

ReagentsVolumeProduct information
mTeSR150mlStem cell technologies, 85850
Recombinant Human BMP-4 Protein5ng/mlR&D systems, 314-BP
Penicillin-Streptomycin (10,000 U/mL)0.5mlThermo Fisher, 15140122
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Jang, J and Kosik, K(2021). Spontaneous and lineage-directed differentiation of hESCs and hiPSCs. Bio-protocol Preprint. bio-protocol.org/prep1247.
  2. Jang, J., Han, D., Golkaram, M., Audouard, M., Liu, G., Bridges, D., Hellander, S., Chialastri, A., Dey, S. S., Petzold, L. R. and Kosik, K. S.(2019). Control over single-cell distribution of G1 lengths by WNT governs pluripotency. PLoS Biology 17(9). DOI: 10.1371/journal.pbio.3000453

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