Figure 1 - Harnessing developmental cues for cardiomyocyte production
Figure 1: Heart size at selected stages of normal cardiac development. (A) Brightfield images capturing murine hyperplasia over E9.5 (whole embryo; dashed white lines indicate the location of the heart), E12.5, E18.5 and P2. Scale bar indicates ~1mm. (B) Brightfield images capturing murine hypertrophy from P13 to P60. Scale bar indicates 5mm. (C) Immunofluorescent images showing pHH3+ cells in E12.5 and ki67+ in LV cells of P2 and P13 hearts. pHH3 and ki67 are proliferation markers and TnT is a cardiomyocyte marker. Abbreviations: Ao, aorta; E, Embryonic days; P, Postnatal days; H, Heart; RA, Right Atrium; LA, Left Atrium, RV, Right Ventricle; LV, Left Ventricle; TnT, Troponin T; pHH3, Phosphohistone H3. Scale bar indicates ~1mm. Figure adapted from (Buikema et al, 2013 & Buikema et al, 2020).
Materials & Methods - Figure 1: Heart size at selected stages of normal cardiac development.
Animal breeding. For animal breeding C57BL/6 (wild-type) mice were used. All animal work was performed within the institutional animal guidelines of Stanford Cardiovascular Institute. Embryonic and fetal wild-type hearts were isolated at embryonic day (E) 9.5, E12.5, and E 18.5. Postnatal wild-type hearts were isolated at day (P) 2, P13, and P60. The tissue was fixated in 4% paraformaldehyde for 30 minutes and transferred to phosphate-buffered saline (pH 7.4). Bright-field microscopy was used to image whole embryos and hearts.
Immunohistochemistry. At selected time points, tissue was snap frozen, embedded in optimal cutting temperature compound, and stored at -80 degrees Celsius. Cryosectioning was performed at a thickness of 10 microns. After 5 minutes of fixation in 4% paraformaldehyde, slides were incubated overnight at 4 degrees Celsius with antibodies for troponin T (TnT), ki67, or phosphorylated Histone H3 (PHH3). After multiple washes slides were incubated for 1 hour at room temperature with selected fluor dyes for the specific primary antibodies. Slides were mounted with glue containing a nuclear stain (DAPI). Regular wide-field fluorescence microscopy was performed to capture images
