How are iCell Cardiac Progenitor Cells cultured and evaluated for differentiation?

iCell® Cardiac Progenitor Cells, which are in transient state and capable of both proliferation and differentiation, have the ability for multilineage differentiation into terminal cardiomyocytes, smooth muscles cells, and endothelial cells (see Drowley et al, 2016). Our optimized Application Protocol for differentiation describes handling iCell Cardiac Progenitor Cells for induction of differentiation by the Wnt inhibitor XAV939 and the activin / TGFβ inhibitor SB431542, analysis of cTNT expression, and basic instructions for culturing the cells for flow cytometry analysis. We recommend running the differentiation protocol immediately upon thawing the cells, and have no data to support other timelines.

The cells do exhibit a basal level of spontaneous proliferation, which increases with the addition of bFGF. Similarly, the cells begin to lose multi-potency upon thaw and will spontaneously differentiate and the Wnt inhibitor directs them toward cardiomyocytes.

Protocol Conditions:

  • Assay ECM: 5 μg/ml fibronectin
  • Assay density: 50,000 cells/well in 96-well plate (156,000 cells/cm2).
  • Assay media: iCell Cardiac Progenitor Cells Maintenance Medium Medium (William’s E Medium, Cocktail B, and gentamicin) + 100 µM XAV939 + 25 µM SB431542
  • Assay timing: Day 6 post-thaw

For more information about markers, see IDENTIFIER 00000146A.

References:

  1. iCell Cardiac Progenitor Cells Prototype User’s Guide
  2. Application Protocol; Modeling Cardiomyocyte Differentiation: Wnt- and Activin/TGFβ-inhibitor Induction with Flow Cytometry Analysis
  3. Presentation (CDI UK Workshop, 2013): iPSC & iPSC derived cells-Values and Applications in drug discovery
  4. Drowley et al (2016) Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation