I am new in the field of iPS cells. I am practicing induction of iPS cells into cardiomyocytes via embryoid bodies. I have difficulties to improve my induction skills. Could you tell me tips and tricks to improve my skills? Thank you.
Salah A. Alshehade
Try to:
- Optimize the formation of embryoid bodies (EBs) - Make sure EBs are of uniform size and shape when initiating differentiation. This can be achieved by carefully pipetting controlled numbers of iPS cells when forming EBs.
- Use cardiogenic growth factors : Adding factors like BMP4, Activin A, and FGF2 during EB formation can enhance cardiac differentiation. Timing and dose is important.
- Monitor differentiation closely : Check EBs frequently for spontaneous beating, which indicates presence of cardiomyocytes. This usually occurs 10-15 days after inducing differentiation.
- Purify cardiomyocytes: Use lactate supplementation or metabolic selection to purify cardiomyocytes away from other cell types after dissociating EBs. This improves purity.
- Optimize dissociation : When dissociating EBs into single cells, try varying enzymatic dissociation times and strengths to maximize viability and yield.
- Assess purity : Use immunostaining (e.g. for troponin T) or qPCR for cardiac marker genes (like MYH6) to assess cardiomyocyte purity. Optimize until >80% purity.
- Culture purified CMs : Mature cardiomyocytes further by culturing for 1-2 weeks post-purification. Optimize culture conditions.
- Assess cardiomyocyte function by looking at beating, electrophysiology, calcium flux, and contractility. Mature, functional CMs should have proper physiology.
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