Fig. 3.

Ex vivo differentiation of HPVCs. a Left panel: After 4 days of BMP2 treatment MESP1-(SSEA1-) cells were collected and cultured in hanging drops. Cells were able to form aggregates, but when plated on collagen I hydrogels they did not adhere or invade the gel. Middle and right panel After SSEA1+ sorting and treatment for 6 days on MEFs with FGF8, FGF2 and VEGF, 40,000 valve progenitors were cultured as hanging drops for 18 h and plated as aggregates on collagen I hydrogels. In 48 h, invasive mesenchymal cells were seen in the gel (−40 µm from the surface), b A total of 20,000 MESP1+ , or MESP1- progenitors or FGF8/FGF2/VEGF-treated MESP1+ progenitor cells were mixed with 20,000 freshly isolated chick H-H stage-24 AV cushion mesenchymal cells to aggregate. Cells were cultured with or without BMP2 for 48 h and stained for post-EMT markers SM alpha actin together with filamin A, periostin, versican, and aggrecan. Very few SSEA1+ and SSEA1+− cells adhered and survived, whereas FGF8/VEGF-treated cells compacted themselves at the middle of the chicken-human cell aggregate and, when triggered with BMP2, some cells started to spread out from the aggregate. c FGF8/VEGF-treated MESP1+ progenitors were placed on the top of E9.5 mouse AVC explants on collagen gels (inset), cultured for 2 days and co-immunostained with anti-human LaminA/C and anti-Sox9, -filamin A or -periostin antibodies. Bar scale indicates 50 μm. Data are representative of at least 6 separate experiments