The analysis of mouse and human embryonic stem cell differentiation cultures has indicated the existence of a cardiovascular progenitor, one of the earliest stages of mesoderm specification to the cardiovascular lineage. In this post, we’ll look, step-by-step, at a new cardiovascular differentiation experimental procedure starting from human pluripotent Stem Cells.
- Yang L. at al. (2008) Nature 453: 524-528
- Kattman S. et al. (2011) Cell Stem Cell 8: 228-240.
Dubois N. et al. (2011) Nature Biotechnology 29: 1011-1018.
Stemgent MesoFate Cardiovascular differentiation protocol
#1 – Embroid Bodies (EB) stage (day 0)
#2- Stage 1 (days 1-3)
Before starting, you’ll need to prepare Induction Medium 1, enough for 2 mL for each well harvested. It is common to observe some debris after 24 hours in culture from a small percentage of cell death during the aggregation process. It is best to remove this debris prior to induction of differentiation. The efficiency of mesoderm induction and cardiovascular progenitor specification in the EBs can be assessed starting at Day 3. This can be monitored by flow cytometric (FC) analysis, evaluating the cells for expression of CD56 and PDGFR-α.
#3- Stage 2 (days 3-5)
Before starting, Induction Medium 2 containing VEGF and IWP2 should be prepared. The efficiency of mesoderm induction and cardiovascular progenitor specific production in the EBs is again assessed at Days 3. This can be monitored by flow cytometric (FC) analysis, evaluating the cells first for expression of CD56 and PDGFR-α followed by KDR and PDGFR-α one day later.
#4- Stage 3 (days 5-12)
Induction is persued using the induction medium 3, containing VEGF. The EBs are cultured in this medium until day 12 (changing medium every 2-3 days as required).
#5- Maintenance (days 12-20)
At this stage the cultivation should be run under normoxic conditions. The Maintenance Medium will be changed every 2-3 days until day 20.
Stain for cardiac specific markers such as CD172 α (Sirp-α) and cardiac-TroponinT at day 20.
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