Dmem f12 hepes

The H14 human embryonic stem cell (hESC) line was obtained from WiCell (Cat No. WAe014-A) and is listed as an NIH-approved line for research purposes (NIH Approval Number: NIHhESC-10–0064). Stem cell culture was done using a commercially available media kit (mTeSR Plus; Cat No. 100–0276, STEM CELL Technologies) following the manufacturer’s published protocol [32 ]. Briefly, cell colonies were grown on Matrigel-coated 6-well plates (Matrigel: Cat No. 354277, Corning; Plates: Cat No. CLS3516–50EA, Sigma Aldrich) in mTeSR Plus for at least three passages before being dissociated for downstream differentiation protocols. Matrigel was diluted according to the lot-specific dilution factor recommended by the manufacturer in 25 ml of DMEM/F12 HEPES (Cat No. 36254, STEMCELL Technologies) and set for 1 hour at 37°C before plates were used. Areas of cellular differentiation were removed each day from cultures. Passaging of colonies was done roughly every five days using Gentle Cell Dissociation Reagent (GCDR) from STEMCELL Technologies (Cat No. 100–0485). Stem cell pluripotency was validated using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies, Cat No. 05230) and human stem cell antibody array from AbCam (Cat No. ab211066) according to the manufacturer’s directions.

The H14 human embryonic stem cell (hESC) line was obtained from WiCell (Cat No. WAe014-A) and is listed as an NIH-approved line for research purposes (NIH Approval Number: NIHhESC-10-0064). Stem cell culture was done using a commercially available media kit (mTeSR Plus; Cat No. 100–0276, STEMCELL Technologies) following the manufacturer’s published protocol [29 ]. Briefly, cell colonies were grown on Matrigel-coated 6-well plates (Matrigel: Cat No. 354277, Corning; Plates: Cat No. CLS3516-50EA, Sigma Aldrich) in mTeSR Plus for at least three passages before being dissociated for downstream differentiation protocols. Matrigel was diluted according to the lot-specific dilution factor recommended by the manufacturer in 25 ml of DMEM/F12 HEPES (Cat No. 36254, STEMCELL Technologies) and set for 1 h at 37 °C before plates were used. Areas of cellular differentiation were removed each day from cultures. Passaging of colonies was done roughly every five days using Gentle Cell Dissociation Reagent (GCDR) from STEMCELL Technologies (Cat No. 100–0485). Stem cell pluripotency was validated using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies, Cat No. 05230) and human stem cell antibody array from AbCam (Cat No. ab211066) according to the manufacturer’s directions.