Fig 2: Figure 3. The amount of secreted Cer1 correlated with the proportion of DE differentiation of mouse ES/iPS cells.(A, B) The proportion of DE cells assayed by flow cytometry correlated with the amount of the Cer1 protein secretions assayed on day (D) 5 (A) or D7 (B) of differentiation using the SK7 ES cell line. Different growth factors were added for obtaining different degrees of definitive endoderm (DE) differentiation. A10, Activin 10 ng/ml; b5, bFGF 5 ng/ml; control (Cont), no growth factors. (C) The proportion of DE assayed by flow cytometry correlated with the level of the secreted mouse Cer1 assayed on D7 of differentiation added with activin and bFGF using the EB3 ES and 20D17 iPS cell lines.

Fig 3: The amount of secreted CER1 correlated with the DE differentiation of human iPS cells.(A) The supernatant was sampled 24 h after replacement with fresh media on differentiation days 1 to 5 (D1 to D5) of the human iPS cell line (253G1). ELISA assays were performed to measure secreted CER1, and quantified by comparison with the standard CER1. The proportion of SOX17+/FOXA2+ DE was calculated from the immunocytochemical analysis results. Values represent the mean ± S.E.M. (N = 3). (B) At D4, approximately all the FOXA2+ cells co-expressed SOX17. (C) Human CER1 staining was observed in approximately all the FOXA2+ cells (D) CER1+ cells did not express T or AFP at D4 in our differentiation system. (E) T is expressed in human iPS cell-derived mesoderm cells. (F) AFP is expressed in human iPS cell-derived hepatic cells (G) ELISA and immunocytechemical analysis of the re-plated DE cells. (H) The proportion of SOX17+/FOXA2+ DE correlated with human CER1 secretion assayed on D4 of differentiation using the 201B7 human iPS cell line and khES3 human ES cell line. Scale bar = 100 µM.

Fig 4: Establishment of an ELISA system for quantification of the secreted mouse Cer1 protein.(A) Schematic drawing of the ELISA system. (B) Standard curve using purified Cer1. (C) ELISA assays for the secreted Cer1 protein at differentiation days (D) 1, D3, D5, or D7. The supernatant was sampled 48 h (lower panel) after replacement with fresh media. Time-dependent expressions of Cer1 in the supernatant, detected by immunoprecipitation, are also shown (bottom panels).

Fig 5: Murine Cerberus1 is a secreted protein expressed in the mouse ES cell-derived definitive endoderm.(A) Cer1 is up-regulated in the definitive endoderm (DE) population and assayed by expression profiling analysis. Y axis: Cer1 expression level; X axis: different germ layer cells and populations on different differentiation days (D) derived from ES cells. ES cells were differentiated into the 3 germ layers under different conditions [12] [25]. ES cell-derived differentiated cells of the ectoderm (ECT) (SSEA1−/Flk1−/PGFRα-), mesoderm (MES) (E-cadherin+/PDGFRα+), paraxial mesoderm (PAM) (E-cadherin−/PDGFRα+/Flk1−), and DE (E-cadherin+/CXCR4+ populations: D5 DE Cxcr4, D7 DE; and E-cadherin+/Daf1+ populations: D5 DE Daf1 [3] at D5 and D7) were prospectively isolated by the expression of specific cell surface antigens using flow cytometry. DE cells at D8 were further subdivided into Pdx1/GFP-negative and -positive populations (D8 DE GFP- and GFP+). (B) Semiquantitative RT-PCR of Cer1, Sox17, Foxa2, Flk1, and Zic1 in ES cell-derived differentiated cells treated with activin A 10 ng/ml and bFGF 5 ng/ml, BMP 7 25 ng/ml, or SB432542 10 µM. (C) Time-dependent expression of Cer1 and Sox17 detected by real-time PCR. (D, E, F) Immunocytochemical analysis. At D7, almost all Foxa2+ cells are co-stained for Sox17 (E) Cer1 is expressed in the Foxa2+ cells (F) Cer1 positive cells do not express T or AFP at D7. (G) T is expressed in mouse ES derived mesoderm cells. (H) AFP is expressed mouse ES derived hepatic cells (I) Western blot analysis for Cer1 with non-reduced/reduced whole cell extract of differentiated ES cells and immunoprecipitation of the supernatant (Sup IP). Scale bar = 100 µM.