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.
Cardiomyocytes generated from hPSCs using MesoFate Differentiation Medium: Tropinin T (red); DAPI (blue).
Human pluripotent stem cell derived Embroid Bodies (EBs) induced with combinations of Activin A, BMP-4, bFGF, VEGF, and Dickkopf homolog 1 (Dkk-1) (or a Wnt pathway antagonist such as IWP2) in serum free media generates a “KDR low/c-Kit neg, PDGFR-α high” cell population leading to cardiomyocytes, endothelial, and vascular smooth muscle cells (1, 3).
The staged protocol described below, and developed in collaboration with the laboratory of Dr. Gordon Keller, describes the cardiovascular differentiation of human pluripotent Stem Cells (hPSCs) using Stemgent MesoFate Differentiation Medium in a 6-well format assay. While this example describes the setup for 3 wells in a 6-well plate, reagent volumes can be adjusted according to the desired number of experimental samples.
Cardiovascular differentiation from day 0 to 12 is carried out under hypoxic conditions, the period between day 12 to 20 is performed under normoxic conditions.
Sources:
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.
The serum free MesoFate media displays high yields of cardiomyocyte differentiation through optimal Embroid body-based mesoderm induction. It supports the differentiation of both induced pluripotent and embryonic stem cells. Additional optimization may be required for scale-up cultures and cardiovascular differentiation from other human pluripotent stem cell types (proliferation rate and differentiation efficiencies varying between cell types).
Schematic representation of the protocol using MesoFate for cardiomyocytes generation. (click to enlarge)
#1 – Embroid Bodies (EB) stage (day 0)
The formation of EBs is the first important step in the differentiation of hPSCs towards cardiovascular lineages and is best achieved by culturing small aggregates of hPSCs in minimal amounts of BMP-4 for 24 hours.
At this stage, BMP-4 functions to promote the survival of the hPSCs. The aggregation protocol below is based on a healthy, starting culture of hPSCs grown on mouse fibroblasts/feeders in 6-well format. Cells should be growing within a density range of 60 -80% confluency (Figure B).
The figure below gives an overview of the experimental set-up and examples of proper density, health, and confluency of starting hPSCs cultures. The instructions should be followed under Hypoxic conditions.
(A) Culture plate experimental set up in tissue culture plate. 3 wells containing hPSCs are used for cardiovascular differentiation, one is used to estimate total cell number. (B) Healthy, feeder-free starting culture of hPSCs grown on mouse fibroblasts/feeders at approximately 80% confluency. (click to enlarge)
#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)
Sirp α & cTnT expression at Day 20 by FC analysis. cTnt & Sirpα+ cardiovascular cells are in red boxes.
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.
Want to benefit from MesoFate Medium’s superior performances ?
Differentiation to cardiomyocytes with the Stemgent MesoFate Differentiation medium results in a reproducible 250% increase of cardiomyocytes relative to input hPSCs, and 1.8 fold more cardiomyocytes than competitor medium, when using the same protocol.
Cardiomyocyte output using MesoFate or Competitor Medium. MesoFate Differentiation Medium generates a greater than 2.5-fold expansion of cardiomyocytes relative to the starting numbers of pluripotent stem cells, significantly higher yield vs. competitor medium.
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![Plating and starting culture of hPSCs. (A) Experimental set up in tissue culture plate. 3 wells containing hPSCs will be used for cardiovascular differentiation while the fourth one is used to estimate total cell number. [B] Healthy, feeder-free starting culture of hPSCs grown on mouse fibroblasts/feeders at approximately 80% confluency. (click to enlarge)](http://beingbioreactive.files.wordpress.com/2014/09/plating-and-starting-culture-of-hpscs.png)
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.
