Generation of mesendoderm using a combination of Activin, FGF2 and BMP4.

<p>(A) Colony morphologies formed in response to the three-step protocol to differentiate hESCs into mesendoderm progeny. H9 cells were grown for 2 days in CDM/PVA+Activin 10 ng/ml+FGF2 12 ng/ml, then for 72 hours in CDM/PVA+SU5402 10 µM+Activin 5 ng/ml. The next 4 days, cells were grown in CDM/PVA+Activin 30 ng/ml+FGF2 20 ng/ml+BMP4 10 ng/ml. Images of the same colonies were captured every day for 9 days. Scale Bar 200 µM. (B) Effect of different combinations and doses of Activin, FGF2 and BMP4 on the differentiation of hESCs grown in CDM/PVA. Following the third day in CDM/PVA+SU5402 10 µM, H9 cells were induced to differentiated in CDM/PVA supplemented with different combination of growth factors. RNAs were extracted after 3 days and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. hESCs grown in CDM+Activin+FGF or differentiated in CDM+SB431542+FGF2 were used as negative controls. (C) Expression of specific markers for mesendoderm in hESCs differentiated in CDM/PVA supplemented with Activin, FGF2 and BMP4 in the three step protocol. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (D) Microarray gene expression heat map to compare human embryonic stem cells (ESC) grown in CDM supplemented with Activin and FGF and mesendoderm cells generated in CDM/PVA supplemented with Activin, FGF and BMP4 (LE). Up-regulation is coloured in shades of red and down-regulation in shades of blue according to the log z scale shown at the bottom of the heat map. Gene names marked with an asterisk denote genes that pass a significant differential regulation threshold. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006082#s4" target="_blank">Material and Methods</a>). (E) Fraction of cells expressing the definitive endoderm marker CXCR4 and the mesendoderm/mesoderm marker PDGFαR after induction of differentiation in CDM PVA in the presence of increasing doses of Activin. H9 cells were differentiated following the three step protocol described above in the presence of Activin (30 or 100 ng/ml). Fraction of cells expressing CXCR4 or PDGFαR was determined using FACS 8 days after plating. (F) RT-PCR analyses for the expression of liver markers (Albumin, HNF4, αFP), gut marker (CDX2) and cardiac markers (ANF, α Actinin, α-1 Channel) in endoderm progenitors grown in media containing serum. Endoderm progenitors generated using the three step protocol were differentiated in media containing three different FBS lots. RNAs were extracted after 5 and 10 days of differentiation and the expression of genes expressed was analysed using RT-PCR.