Vascular smooth muscle cells (VSMCs) play a pivotal role in the regulation of blood vessel function and are implicated in various cardiovascular diseases. Creative Biolabs offers a comprehensive protocol for differentiating stem cells into distinct VSMC subtypes. This protocol provides detailed instructions for the materials required, procedure, and important notes to ensure successful differentiation.
Thaw and culture pluripotent stem cells in a suitable culture medium, maintaining pluripotency. Passage the cells as needed to maintain an undifferentiated state.
For ESCs or iPSCs, switch to a basal medium with reduced serum concentration (2-5%) to initiate differentiation towards the mesodermal lineage. Add retinoic acid (RA) at a concentration to promote mesodermal specification. Culture for 24-72 hours.
Remove RA and replace with a basal medium containing PDGF and TGF-β. Incubate for 7-14 days with medium changes every 2-3 days. Monitor cell morphology under the microscope for the appearance of spindle-shaped cells characteristic of VSMCs.
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.
Confirm VSMC differentiation through immunocytochemistry, flow cytometry, and gene expression analysis of VSMC markers (e.g., α-smooth muscle actin, calponin, SM22α). Assess VSMC contractility, proliferation, and migration capabilities as per experimental requirements.
Creative Biolabs remains committed to supporting cutting-edge stem cell research, and we encourage researchers to adapt and optimize this protocol to meet their unique needs. The resulting VSMCs can be used for various applications, including tissue engineering and disease modeling.
We are committed to providing high-quality protocols and reagents to advance stem cell research. For more information, please contact us.
References
For Research Use Only. Not For Clinical Use.
