Stem cell differentiation is a powerful tool in developmental biology and disease modeling. Extraembryonic endoderm cells play a crucial role in early embryonic development and have the potential to differentiate into various cell types, including those of the gastrointestinal and respiratory tracts.
This protocol outlines the steps for differentiating stem cells into extraembryonic endoderm cells, a process that holds great promise for advancing our understanding of early development and for therapeutic applications. At Creative Biolabs, we are committed to providing cutting-edge protocols for stem cell research.
Culture pluripotent stem cells in Matrigel/Gelatin-coated plates using pluripotent stem cell medium. Maintain the pluripotent stem cells in an undifferentiated state by changing the medium daily, and passage them as needed.
When pluripotent stem cells reach 70-80% confluency, wash the cells with PBS. Replace the pluripotent stem cell medium with RPMI 1640 medium. Add Activin A to the RPMI medium and incubate for 24 hours. During this period, cells will undergo mesendoderm induction.
Replace the differentiation medium with fresh differentiation medium without Activin A but supplemented with Wnt3a and FGF2. This promotes definitive endoderm formation. Change the differentiation medium to one containing BMP4 and basic FGF. This step encourages the differentiation of definitive endoderm into extraembryonic endoderm cells.
Continue culture in basal medium supplemented with PDGF and TGF-β, adjusting the concentrations as needed. For subtype specification, tailor the growth factor cocktail accordingly.
Continue to replace the differentiation medium with BMP4 and basic FGF every 2-3 days until extraembryonic endoderm cells mature, typically around day 10-12. Monitor cell morphology under the microscope for characteristic changes. Fix and stain cells with specific markers to confirm their differentiation into extraembryonic endoderm cells.
This protocol serves as a foundational guide, but researchers are encouraged to adapt and optimize the procedure based on their unique experimental needs and cell lines. Creative Biolabs is committed to supporting your research efforts and can provide expert consultation and high-quality services to facilitate your stem cell differentiation studies.
In case you need any further assistance or specialized services for your stem cell differentiation experiments, please do not hesitate to contact us. We are here to help you advance your research and contribute to the exciting field of stem cell biology.
Reference
For Research Use Only. Not For Clinical Use.
