Hedgehog signalling-an essential pathway during embryonic pancreatic development, the misregulation of which has been implicated in several forms of cancer-may also be an important mediator in human pancreatic carcinoma [1][2][3][4][5][6][7][8] . Here we report that sonic hedgehog, a secreted hedgehog ligand, is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions: pancreatic intraepithelial neoplasia (PanIN). Pancreata of Pdx-Shh mice (in which Shh is misexpressed in the pancreatic endoderm) develop abnormal tubular structures, a phenocopy of human PanIN-1 and -2. Moreover, these PanIN-like lesions also contain mutations in K-ras and overexpress HER-2/neu, which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signalling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signalling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. These data suggest that this pathway may have an early and critical role in the genesis of this cancer, and that maintenance of hedgehog signalling is important for aberrant proliferation and tumorigenesis.Sonic hedgehog (SHH) is misexpressed in human adenocarcinoma and its precursor lesions. SHH expression was determined using in situ hybridization to detect SHH messenger RNA and immunohistochemistry (IHC) to detect the protein with an antibody directed against Competing interests statementThe authors declare that they have no competing financial interests. . Pancreatic tissues were obtained from 20 specimens resected for pancreatic cancer. Control pancreatic tissues with no evidence of abnormality or autolysis upon histological evaluation were obtained from autopsy specimens or from pancreatic resections for trauma. In normal adult human pancreata, no SHH was detected in the islets, acini or ductal epithelium (Fig. 1a). However, evaluation of pancreata from patients with adenocarcinoma reveals that SHH is aberrantly expressed in 70% of specimens. Normal ductal epithelium does not express detectable levels of SHH (Fig. 1b); however, as the ductal epithelium shows increasing degrees of atypia, PanIN-1 to -3 ( Fig. 1c-e), a higher expression of SHH is observed. SHH expression is also detected in the malignant epithelium of adenocarcinoma samples (Fig. 1f). This expression pattern was also confirmed by our in situ hybridization for SHH mRNA ( Supplementary Fig. 1). NIH Public AccessLoss of regulation in this pathway has been implicated in several human cancers 10,11 . Thus in order to determine the potential role of SHH misexpression in the adult human pancreas, pancreata from transgenic mice (gift of H. Edlund) in which Shh misexpression was driven by the pancreatic-specific Pdx-1 promoter were histologically and immunohistochemically analysed.A total of four pancreata from three-week-old Pdx-Shh mice were histologically evaluated by a gastro...

Summary Tumors are largely classified by histological appearance, yet morphological features do not necessarily predict cellular origin. To determine the origin of pancreatic ductal adenocarcinoma (PDA), we labeled and traced pancreatic cell populations after induction of a PDA-initiating Kras mutation. Our studies reveal that ductal and stem-like centroacinar cells are surprisingly refractory to oncogenic transformation, whereas acinar cells readily form PDA precursor lesions with ductal features. We show that formation of acinar-derived premalignant lesions depends on ectopic induction of the ductal gene Sox9. Moreover, when concomitantly expressed with oncogenic Kras, Sox9 accelerates formation of premalignant lesions. These results provide insight into the cellular origin of PDA and suggest that its precursors arise via induction of a duct-like state in acinar cells.

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Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1 + and subsequent PDX1 + /NKX6.1 + pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1 + /NKX6.1 + progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell *

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-␤B and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.

Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports suggest that there may be important differences between them. We performed a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low passage iPS cells analyzed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 reduces the efficiency of human iPS cell generation, suggesting that somatic memory genes may be functionally relevant during reprogramming.