Refer to the product datasheet for complete product details.
Briefly, the pluripotency status of human stem cells is verified using the following in vitro differentiation procedure:
- Culture pluripotent cells of interest
- Induce endoderm, mesoderm, and ectoderm differentiation with media supplements
- Evaluate differentiation using germ layer markers and fluorescent ICC
Reagents supplied in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B):
The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into hepatocyte-like cells.
Other Supplies Required
- BSA, very low endotoxin
- D-MEM/F-12 (1X)
- GlutaMAX™ (Invitrogen or equivalent)
- Phosphate Buffered Saline (PBS)
- Trypan Blue Solution
- MEF Conditioned Media (Catalog # AR005 or equivalent)
- StemXVivo™ Culture Matrix (100X) (Catalog # CCM013), Cultrex® PathClear® BME Reduced Growth Factor Basement Membrane Extract (Catalog # 3433-005-01), or equivalent
- Recombinant Human FGF basic (Tissue culture grade; Catalog # 4114-TC or equivalent)
- Accutase® (Innovative Cell Technologies or equivalent)
- Human pluripotent stem cells
- 24-well culture plates
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 0.2 μm syringe filter
- 10 mL syringe
- Pipettes and pipette tips
- Serological pipettes
- 37 °C and 5% CO2 incubator
- Inverted microscope
- 37 °C water bath
This protocol is designed for BG01V human embryonic stem (hES) cells and iPS2 human induced pluripotent stem (iPS) cells grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005). If using different cell lines or growth media, this protocol may need to be optimized.
Note: The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.
Undifferentiated Cell Preparation
Add 1:100 Culture Matrix or Cultrex BME in sterile PBS.
Add 1.1x105 cells/cm2 in MEF Conditioned Media containing 4 ng/mL FGF Basic.
| || |
|Day 1 ||Replace media with Ectoderm Differentiation Media. ||Replace media with |
Mesoderm Differentiation Media.
|Replace media with Endoderm Differentiation Media. |
|Day 2 ||Repeat ||Repeat media change 12-16 hours later. |
ICC detection of Brachyury (24-36 hours after initial differentiation)
|16-24 hours later, Replace media with Endoderm Differentiation Media II. |
|Day 3 ||Repeat || ||Replace media with Endoderm Differentiation Media II. |
|Day 4 ||ICC detection of Otx2. || ||ICC detection of SOX17. |