Supplementary Figure 4: Testing nEnd potency by chimera injection.
From: Insulin fine-tunes self-renewal pathways governing naive pluripotency and extra-embryonic endoderm
(A) Schematic of injection experiment, indicating the process for preparing the injected cells with a brief accutase wash, and the potential locations where injected H2B-mCherry positive cells could contribute in late blastocysts (Epiblast, PrE and trophoblast) and E6.5 (epiblast, visceral endoderm, parietal endoderm). (B) Flow cytometry for PDGFRA and mCherry during HVMC nEnd adaptation to nEnd culture conditions. Cells were passaged in preparation for blastocyst injection, and maintained a high level of PDGFRA and H2B-mCherry expression. Numbers indicate the percentage of PDGFRA and mCherry double positive cells. Dot plots are representative of 3 HVMC passages. (C) Confocal sections showing contribution to both visceral and parietal endoderm. Data supports the images and experiment shown in Fig. 4f. Each row shows a pair of photos in different optical planes, from three different embryos with contribution in both Reichert’s membrane and the visceral endoderm. Images show confocal optical sections, with overlaid images from the brightfield, red (H2B-mCherry) and green (autofluorescence) fluorescent channels. (D) Confocal sections showing three embryos with chimeric contribution to only the visceral endoderm (top photo), or Reichert’s membrane (lower two photos, shown partially dissected). Images show confocal optical sections, with overlaid images from the brightfield, red (H2B-mCherry) and green (autofluorescence) fluorescent channels.
