Albumin induced in airway progenitors derived from Mgp^−/− ESCs when co-cultured with Mgp^−/− ECs. (a) Schematic diagram of the experimental strategy. KDR, kinase insert domain receptor. (b) Flow cytometric analysis of EpCAM⁺c-kit⁺ and CXCR4⁺c-kit⁺ cell populations in definitive endoderm (DE) derived from WT (Mgp^+/+) and Mgp^−/− ESCs (n = 3). (c) Gene expression of lineage markers was determined by real-time PCR with normalization to GAPDH expression in airway progenitors derived from WT (Mgp^+/+) and Mgp^−/− EpCAM⁺c-kit⁺ definitive endoderm cells. Fold changes of expression are calculated as compared with the FoxN1 expression in Mgp^+/+EpCAM⁺c-kit⁺ definitive endoderm cells (n = 3). (d) Expression of von Willebrand factor (vWF), CD31, and VE-cadherin in induced ECs derived from Mgp^+/+ and Mgp^−/− ESCs. The C166 line is shown as control (n = 3). (e) Expression of EGFP driven by the Flk1 promoter in C166 cells and Mgp^+/+- and Mgp^−/−-induced ECs (n = 5). (f) Time course of MGP expression during the derivation of EC and definitive endoderm from Mgp^+/+ and Mgp^−/− ESCs (n = 3). (g) Gene expression of HGF, Flt1, and c-Met was determined by real-time PCR in airway progenitors (AP) derived from Mgp^+/+ and Mgp^−/− EpCAM⁺c-kit⁺ definitive endoderm cells (n = 3). (h) Gene expression of HGF, Flt1, and c-Met was determined by real-time PCR in induced ECs derived from Mgp^+/+ and Mgp^−/− ESCs (n = 3). (i) Albumin expression was determined by real-time PCR in Nkx2.1⁺FoxP2⁺ cells derived from Mgp^+/+ and Mgp^−/− EpCAM⁺c-kit⁺ definitive endoderm cells with co-culture of Mgp^+/+ and Mgp^−/− ECs. Anti-Flt1 (a-Flt1) and anti-HGF (a-HGF) were added to neutralize Flt1 and HGF, respectively (n = 3). (j) EGFP and mCherry were determined in the co-culture of airway progenitors and ECs by fluorescence microscopy (n = 4). BF, bright field. (k) Gene expression of HGF, Flt1, and c-Met was determined by real-time PCR in C166 ECs transfected with scrambled siRNA (SCR) or MGP siRNA (si) (n = 3). (l) Albumin expression was determined by real-time PCR in Nkx2.1⁺FoxP2⁺ cells derived from Mgp^+/+ and Mgp^−/− EpCAM⁺c-kit⁺ definitive endoderm cells with co-culture of MGP-depleted C166 ECs. a-Flt1 and a-VEGF were added to neutralize Flt1 and VEGF, respectively (n = 3). (m) EGFP and mCherry were determined by fluorescence microscopy in airway progenitors derived from Mgp^−/− EpCAM⁺c-kit⁺ definitive endoderm cells with co-culture of C166 ECs (n = 5). Data in c and d, f–i, and k and l were analyzed by two-sided t test. **, P < 0.01; ***, P < 0.001. Error bars are standard deviation. Data distribution was assumed to be normal, but this was not formally tested. Bars, 50 µm.