Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease

Abstract

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 Nkx2.5 R141C/+ 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 Nkx2.5 R141C/+ 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. C 2018 by John Wiley & Sons, Inc.Keywords: cardiac differentiation r embryoid bodies r embryos r hanging drops r mouse embryonic stem cells

Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression

“…Phase-contrast microscopy, immunofluorescence staining, and qPCR analysis were performed as in [12]. Cell counting for Hoechst (Sigma–Aldrich, St. Louis, USA, B-2883) and T (Abcam, Toronto, Canada, AB20680) nuclear staining was performed as in [129]. Primary antibodies against PAX7 (Developmental Studies Hybridoma Bank, Iowa City, USA, AB528428), MKI67 (KI67)(Abcam, AB15580), MYOD1 (Abcam, AB133627), and secondary Cy3 goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, USA, 111-165-003), AlexaFluor546 goat anti-mouse IgG1 (ThermoScientific, Waltham, USA, A21123), and AlexaFluor647 goat anti-rabbit IgG (ThermoScientific, A21244) antibodies were also used.…”