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Cell Biology JoVE (Open Access) Citable · DOI

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

DOI: 10.3791/2656-v
What you'll learn
  • ✓ Generate embryonic bodies from mouse ES cells using 96-well standardized format
  • ✓ Identify critical time windows for Wnt/β-catenin and BMP signaling in cardiogenesis
  • ✓ Quantify cardiogenic induction efficiency using a reliable ES cell-based assay
  • ✓ Apply methodology to study other cell lineage differentiation pathways
Protocol

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.

Difficulty
advanced
Total time
~4–7 days (EB formation ~3–5 days; signaling pathway interrogation ~2–3 days)
Model organism
Mouse embryonic stem cells (ES cells)
Biosafety
BSL-1

Steps

1
Form embryonic bodies in 96-well plates

Culture mouse ES cells in round-bottom 96-well microtiter plates to generate uniform embryonic bodies. This standardized format provides reliable and reproducible cardiogenic differentiation.

▶ 01:30
2
Identify critical signaling pathway time windows

Apply targeted Wnt/β-catenin and BMP pathway modulation at different time points during EB differentiation to map essential activation windows for cardiogenic induction.

▶ 03:24
3
Verify time windows using hanging droplet method

Validate identified critical time windows by repeating the signaling pathway interrogation in hanging droplet-derived EBs to confirm reproducibility across EB generation methods.

▶ 05:04
4
Determine critical time points for cardiogenesis

Quantify cardiogenic efficiency at validated time points using morphological and molecular markers to establish definitive critical windows for ES cell cardiogenic differentiation.

▶ 07:06
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