Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing 'pancreatospheres' in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas

Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of <em>Pseudomonas aeruginosa</em> Keratitis in a Mouse Model

<div><p>Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (<em>Lum</em>^−/−) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that <em>in</em><em>vitro Lum</em>^−/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the <em>Lum</em>^−/− mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in <em>Lum</em>^−/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of <em>Lum</em>^−/− mice compared to wild types. Infected <em>Lum</em>^−/− corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24–48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to <em>Lum^+/−</em> mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the <em>Lum</em>^−/− compared to <em>Lum</em>^+/− infected corneas by 2–5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.</p> </div

Differential regulation of CXCL1 in infected <i>Lum ^+/−</i> and <i>Lum ^−/−</i> corneas.

<p>CXCL1 measured by ELISA increased 24 hrs after infection in both genotypes, with its level being consistently lower in the <i>Lum</i>^−/− corneal extracts - the difference being statistically significant at the 2 d.p.i time point (A). Three hours after infection CXCL1 levels were comparably low in both genotypes (n = 3) (B). The <i>Cxcl1</i> transcript measured by qRT-PCR in quadruplicates/animal was increased over baseline 6 hrs post infection; by 24–48 hrs post infection <i>Lum </i>^−/− corneas showed higher levels of <i>Cxcl</i>1 compared to <i>Lum </i>^+/− (C). * <i>p</i>≤0.05.</p

Increased lumican expression in <i>Lum ^+/−</i> corneas 6 hr post infection.

<p>Lumican transcript relative to18S RNA was significantly increased in <i>Lum</i>^+/− corneas 6 hrs after infection as determined by qRT-PCR (n = 4). * <i>p</i>≤0.05.</p

Poor clearance of <i>P.aeruginosa</i> in infected <i>Lum ^−/−</i> corneas.

<p>Viable bacterial CFU were quantified from infected <i>Lum ^+/−</i> and <i>Lum</i>^−/− corneas. CFU were higher in ocular surfaces sampled with filter lifts (A) and whole eye homogenates (B) during the infection. Both methods yielded significantly higher CFU counts from <i>Lum ^−/−</i> corneas as shown at day 4 (cornea surface lift) and day 2 (whole eye homogenate). *<i>p</i>≤0.05.</p

Proinflammatory cytokines measured by ELISA in <i>P.aeruginosa</i> infected corneas.

<p>TNF-α level was low and comparable in <i>Lum</i>^+/− and <i>Lum</i>^−/− corneal protein extracts 3 hrs after infection (A) but significantly higher in <i>Lum</i>^−/− infected corneas 2 days after infection (B). IL-1β was induced comparably in <i>Lum</i>^+/− and <i>Lum</i>^−/− infected corneas for up to 5 days after infection (C) and IL-12 (D) was detected at very low levels in infected corneas of both genotypes. * <i>p</i>≤0.05.</p

Increased tissue damage in infected corneas of <i>Lum</i>^+/− and <i>Lum ^−/−</i> mice.

<p>Paraffin-embedded sections of eyes 24 hrs (A) and 48 hrs (B) after infection were stained with H and E. To examine tissue damage in mice with comparable disease all infected animals used for histology had an initial disease score of 2 to 3 and showed PMN infiltrations in the cornea and anterior chamber. The <i>Lum ^−/−</i> infected corneas showed large areas of epithelial ulcerations (arrow) and stromal damage (arrowhead). Scale bar, 100 µm.</p

Increased clinical score in <i>P.aeruginosa</i> infected <i>Lum</i>^−/− mice.

<p>Mice were infected with 2×10⁴ CFU <i>P.aeruginosa</i> ATCC19660 in one eye and scored in a blinded manner for 6 days. A representative image of infected eyes 1 and 6 days post infection (d.p.i) shown for each genotype indicates relatively similar severity on day 1 but increased opacity and damage in the <i>Lum</i>^−/− cornea on 6 d.p.i (A). Daily disease scores (B) of individual animals shows increased average scores for infected <i>Lum</i>^−/− corneas.* <i>p</i>≤0.05.</p

<i>Dicer</i> Is Required for Maintenance of Adult Pancreatic Acinar Cell Identity and Plays a Role in Kras-Driven Pancreatic Neoplasia

<div><p>The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted <i>Dicer</i> specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that <i>Dicer</i> is essential for the maintenance of acinar cell identity. Acinar cells lacking <i>Dicer</i> showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to <i>Dicer</i> gene dosage. Homozygous <i>Dicer</i> deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous <i>Dicer</i> deletion accelerated PanIN initiation, revealing complex roles for <i>Dicer</i> in the regulation of both normal and neoplastic pancreatic epithelial identity.</p></div