Figure 6. Cell Fate Determination on PTK7− and PTK7+ populations.

Feeder-free H9 (differentiated for 2 days) were sorted into PTK7+ and PTK7− populations. The two populations were re-aggregated as described in Materials and Methods, and replated onto Matrigel-coated coverslips. The PTK7− and PTK7+ re-aggregates were cultured in PSC media for 2 days, and then differentiated using established protocols. (A) Epithelial and mesenchymal marker analysis in PTK7− and PTK7+ re-aggregates 3 day post-sorting. PTK7+ cells that underwent developmental EMT possessed plasticity to revert back to an epithelial cell type. Undifferentiated H9 were plated on coverslips and fixed 4 days after passage. PTK7− and PTK7+ re-aggregates were plated on coverslips and fixed after 2 days in culture. Undifferentiated H9 (top row), PTK7− re-aggregate (mid row) and PTK7+ re-aggregate (bottom row) were stained for PTK7 (green), epithelial maker (E-CADHERIN, red) and mesenchymal marker (VIMENTIN, white). The 4^th column shows the merged images from three fluorescent channels and DAPI. (B) PTK7+ and PTK7− re-aggregates exhibited comparable definitive endoderm formation. Feeder-free H9, PTK7− and PTK7+ re-aggregates were induced to form definitive endoderm with ActivinA treatment as described. Coverslips were fixed and immunostained with endodermal markers EOMES (red) and SOX17 (green). The 3^rd column shows the merged images from two fluorescent channels and DAPI. (C) PTK7− and PTK7+ re-aggregates exhibited comparable neural differentiation. XFiPSC2, PTK7− and PTK7+ re-aggregates were induced to form neural rosettes as previously described. Coverslips were fixed and immunostained with neural markers SOX1 (red), SOX2 (green) and NESTIN (white). The 4^th column shows the merged images from three fluorescent channels and DAPI. All images taken at 10X.