brachyury locus, we demonstrate that endoderm develops from a brachyury + population that also displays mesoderm potential. Transplantation of cells generated from activininduced brachyury + cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

Purpose:The programmed death-1ligand/programmed death-1 (PD-L/PD-1) pathway has been recently suggested to play a pivotal role in the immune evasion of tumors from host immune system. In this study, we tried to reveal the clinical importance and therapeutic potential of the PD-L/ PD-1pathway in pancreatic cancer, which is one of the most aggressive and intractable malignant tumors. Experimental Design: We used immunohistochemistry to investigate PD-L expression in 51 patients with pancreatic cancer who underwent surgery and explored the therapeutic efficacy of blocking the PD-L1/PD-1pathway in murine pancreatic cancer in vivo. Results: PD-L1^positive patients had a significantly poorer prognosis than the PD-L1^negative patients, whereas there was no significant correlation of tumor PD-L2 expression with patient survival. PD-L1 expression was inversely correlated with tumor-infiltrating T lymphocytes, particularly CD8 + T cells. These clinical data have suggested that the PD-L1/PD-1 pathway may be a critical regulator in human pancreatic cancer. Monoclonal antibodies against PD-L1 or PD-1 induced a substantial antitumor effect on murine pancreatic cancer in vivo. PD-L1 blockade promoted CD8 + T-cell infiltration into the tumor and induced local immune activation. Furthermore, the combination of anti^PD-L1 monoclonal antibody and gemcitabine exhibited a significant synergistic effect on murine pancreatic cancer and resulted in complete response without overt toxicity. Conclusion: Our data suggest for the first time that PD-L1 status may be a new predictor of prognosis for patients with pancreatic cancer and provide the rationale for developing a novel therapy of targeting the PD-L/PD-1pathway against this fatal disease.

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP -Flk1 -, GFP + Flk1 -and GFP + Flk1 + cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.

When differentiated in the presence of activin A in serum-free conditions, mouse embryonic stem cells efficiently generate an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45-70%) for cells that express the alpha-fetoprotein and albumin proteins. These cells also express transcripts of Afp, Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store glycogen, show ultrastructural characteristics of mature hepatocytes, and are able to integrate into and proliferate in injured livers in vivo and mature into hepatocytes expressing dipeptidyl peptidase IV or fumarylacetoacetate hydrolase. Together, these findings establish a developmental pathway in embryonic stem cell differentiation cultures that leads to efficient generation of cells with an immature hepatocytic phenotype.