How to induce pluripotent stem cells in to definitive endoderm
In this video, you will see how to generate definitive endoderm cells from PSCs using the Gibco PSC Definitive Endoderm Induction Kit. The Gibco PSC Definitive Endoderm Induction Kit is a complete, ready-to-use media system for efficient induction of pluripotent stem cells to definitive endoderm lineage in 2 days. You can access the product insert and the quick reference protocol online. A supplemental list of reagents used in this protocol can also be referenced here. First, coat the plate with prepared vitronectin solution. Thaw vitronectin at room temperature and dilute with PBS. After dilution, add 1 mL to each well of a 6-well plate. Incubate at room temperature for 1 hour. Then, you'll need to prepare complete Essential 8 Medium. Find details for this preparation on the web link listed below. Next, prepare a cell recovery solution by adding 250 µL of RevitaCell Supplement to 25 mL of Essential 8 Medium. Let media warm to room temperature. When you're ready to seed cells aspirate the vitronectin solution from the coated plate and add 1 mL of cell recovery solution to each of the wells. When PSC culture has reached 70% confluence, cells are ready to be seeded for induction to definitive endoderm. Remove spent Essential 8 Medium and rinse each well with PBS. Add 1 mL Accutase Cell Dissociation Reagent to the well and incubate for 5 minutes or until the colonies freely come into suspension. Collect cell clumps in a 15 mL tube containing 8 mL of Essential 8 Medium. Wash off well with an additional 1 mL of Essential 8 Medium to collect leftover cells. Centrifuge the cells at 200 x g for 5 minutes. After centrifugation, aspirate the supernatant. Flick the tube to dislodge cells and resuspend cells in 10 mL of cell recovery solution prepared previously. Seed PSC clumps at about 1:10 split ratio into vitronectin-coated plates so that 15 to 30% confluence is achieved by day 1. It is suggested that you perform a range-finding study prior to starting definitive endoderm induction to identify optimal seeding density, which will achieve 15 to 30% confluence by day 1. Thaw Definitive Endoderm Induction Medium A to room temperature. Shake the bottle several times to ensure even distribution of the components in the medium. If the cells are 15 to 30% confluent, proceed with induction. Completely aspirate spent Essential 8 Medium from the wells and add pre-warmed Definitive Endoderm Induction Medium A. Thaw Definitive Endoderm Induction Medium B to room temperature. Shake the bottle several times to ensure even distribution of the components. Completely aspirate spent medium from the wells and add pre-warmed Definitive Endoderm Induction Medium B. After 24 hours, cells are ready to be assayed for definitive endoderm characteristics or further differentiated to downstream lineages. You might observe some floating cells at this stage. These can be easily removed by aspiration without being detrimental to the cells.