We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.
Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.
1. Embryonic Body (EB) formation using 96-round bottom well microtiter plates
2. Identification of critical time windows of the key signaling pathway(s) for cardiogenesis
3. Verification of time windows of signaling for cardiogenesis in EBs made from hanging droplets
The hanging drop method has been the conventional method used for EB formation and in vitro differentiation. However, it is laborious and limits experimental flexibility due to logistical concerns. By the same token, results are also more difficult to validate as the skill of the experimenter is crucial to successful EB formation and manipulation as hanging drops. A simpler method is to form EBs in a round-bottomed 96-well plate as a single-step process. This format allows in vitro differentiation to be standardized and can accommodate more experimental conditions due to the ease of EB formation and downstream manipulation (e.g. modulator addition or removal). Furthermore, the 96-well plate format can be optimized to be an efficient screening tool in mouse ES cells.
The authors have nothing to disclose.
This work was supported by Veterans Affairs and NIH grants 5U01HL100398 and 1R01HL104040.