We have established an in vitro model of the human congenital heart defect (CHD)-associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. © 2018 by John Wiley & Sons, Inc.
Keywords: cardiac differentiation; embryoid bodies; embryos; hanging drops; mouse embryonic stem cells.
© 2018 John Wiley & Sons, Inc.