H9 human embryonic stem cells hescs

Matrigel-coated plates were prepared by resuspending a frozen 2.5-mg aliquot of Matrigel, Growth Factor Reduced (Corning, Glendale, AZ) in 1 ml of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Life Technologies, Carlsbad, CA) and diluting the resulting solution in 29 ml of DMEM/F12. One milliliter of this solution was used to coat each well of five six-well plates. Plates were stored at 37°C for at least 1 hour before use and up to 1 week. The following hPSC lines were used: H9 human embryonic stem cells (hESCs) (81 (link)) (WiCell, Madison, WI), IMR90-4 induced pluripotent stem cells (iPSCs) (82 ) (WiCell), DF19-9-11T iPSCs (83 (link)) (WiCell), and WTC11 iPSCs (84 (link)) (Gladstone Institutes, San Francisco, CA). hPSCs were maintained at 37°C, 5% CO₂ on Matrigel-coated plates in E8 medium (STEMCELL Technologies, Vancouver, Canada) with daily medium changes. When hPSC colonies began to touch, cells were dissociated as colonies using ~7 min of Versene (Life Technologies) treatment and transferred to a new Matrigel-coated plate at a split ratio of 1:12.