How are iCell Cardiac Progenitor Cells cultured and evaluated for proliferation?
iCell® Cardiac Progenitor Cells, which are in transient state and capable of both proliferation and differentiation, can be cultured directly in HTS plates making them amenable to extensive high content analysis of proliferation cells (see Drowley et al, 2016). Our Application Protocol for modeling cardiac proliferation has demonstrated utility in inducing proliferation of iCell Cardiac Progenitor Cells with basic fibroblast growth factor (bFGF), assessed by the expression of NKX2.5 using high content analysis. We recommend running the proliferation 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 (a 1.6-fold increase over 2 days). Similarly, the cells begin to lose multi-potency upon thaw and will spontaneously differentiate and the Wnt inhibitor directs them toward cardiomyocytes.
We have not extensively characterized the proliferation rate of these cells, but in some protocols we have seen them double every 24 hours for the first 2 to 3 days.
- Assay ECM: 5 μg/ml fibronectin
- Assay density: 25,000 cells/well in 96-well plate (78,000 cells/cm2)
- Assay media: iCell Cardiac Progenitor Cells Maintenance Medium (William’s E Medium, Cocktail B, and gentamicin) + 1 μg/ml bFGF
- Assay timing: Day 2 post-thaw
- iCell Cardiac Progenitor Cells Prototype User’s Guide
- Application Protocol: Modeling Cardiac Proliferation: bFGF Induction with High Content Analysis
- iPSC & iPSC Derived Cells - Values and Applications in Drug Discovery
- 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