H9 and H1 human embryonic stem cells (hESC; NIH code: WA09 and WA01,
respectively) were initially grown on mitotically inactivated primary mouse
embryonic fibroblasts (MEF) in KnockOut DMEM/F12 supplemented with 20% KnockOut
serum replacement (KSR), 0.1 mM β-mercaptoethanol, and non-essential
amino acids (Gibco). Adaptation to single-cell based non-colony type monolayer
culture of hESC was carried out as previously described [41 (link)–43 (link)].
Briefly, the cell pellets of hESC were dissociated with 1× Accutase
(Innovative Cell Technologies) for 15 min and subsequently resuspended in mTeSR1
defined medium (Stemcell Technologies), and centrifuged at 2000 rpm for 5 min.
Dissociated single cells were then plated at a density of 2 ×
106 cells in 6-well dishes coated with 2.5% hESC-qualified
Matrigel (Corning) in mTeSR1 containing 10 μM ROCK inhibitor (Tocris
Bioscience). After 24 h, the ROCK inhibitor was withdrawn and hESC were allowed
to grow as a monolayer. hESC were passaged every 4–5 days, replated at a
dilution of 1:6 and maintained in mTeSR1 in Matrigel-coated culture dishes.
respectively) were initially grown on mitotically inactivated primary mouse
embryonic fibroblasts (MEF) in KnockOut DMEM/F12 supplemented with 20% KnockOut
serum replacement (KSR), 0.1 mM β-mercaptoethanol, and non-essential
amino acids (Gibco). Adaptation to single-cell based non-colony type monolayer
culture of hESC was carried out as previously described [41 (link)–43 (link)].
Briefly, the cell pellets of hESC were dissociated with 1× Accutase
(Innovative Cell Technologies) for 15 min and subsequently resuspended in mTeSR1
defined medium (Stemcell Technologies), and centrifuged at 2000 rpm for 5 min.
Dissociated single cells were then plated at a density of 2 ×
106 cells in 6-well dishes coated with 2.5% hESC-qualified
Matrigel (Corning) in mTeSR1 containing 10 μM ROCK inhibitor (Tocris
Bioscience). After 24 h, the ROCK inhibitor was withdrawn and hESC were allowed
to grow as a monolayer. hESC were passaged every 4–5 days, replated at a
dilution of 1:6 and maintained in mTeSR1 in Matrigel-coated culture dishes.
