Fig. 4.

In vivo differentiation of HPVCs in mouse embryonic heart. SSEA1+ MESP1+ Sox9GFP cells were sorted and treated with VEGF/FGF8 for 7 days before injection into E10.5 mouse AVC. a An E10.5 embryo is stretched to expose the AVC. Dye injection shows the target of the injection. Scale bars are 1 mm. b Two hours after injection the hearts were dissected and placed in culture for two days on Matrigel coated inserts (top inset). The heart sections were immunostained with anti-human LmnA/C, –GFP, and –Sox9 antibodies. c Two sites of injection and resulting cell fate. Cells were injected into the AVC or the chamber. Only the cells injected into the AVC (visualized on the eosin/hematoxylin stained heart section in the inset) underwent EMT and expressed Sox9-GFP. d, e Sox9GFP VEGF/FGF8-treated cells were injected into the AVC and the hearts were cultured for two days (d) or the whole embryos were cultured in their yolk sac for 36 h (e).The heart sections were stained with anti-filaminA, -periostin or –collagen antibodies. The middle panels in (d) show the AVC region and the right panels a high magnification of a subset of cells within the AVC (e). A section of the whole embryo is shown in the left panel; the middle and right panels show the heart and the cells injected in the AVC. The heart sections were stained with anti-periostin antibody. Results are representative of at least 9 experiments. SAC superior AV cushions, IAC inferior AV cushions, PLV primary left ventricle, A atrium. The scale bars in b-e indicate 50 μm