Identification of a candidate stem cell in human gallbladder.

There are currently no reports of identification of stem cells in human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13- cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells.

Polymerase η suppresses telomere defects induced by DNA damaging agents.

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη's ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.

Human bone marrow-derived mesenchymal stem cells display enhanced clonogenicity but impaired differentiation with hypoxic preconditioning.

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O(2) consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.

Amnion-derived multipotent progenitor cells inhibit blood monocyte differentiation into mature dendritic cells.

Cells derived from the placenta have become the focus of extensive research concerning their ability to be used for regenerative medicine or cellular therapies. In a previous study, we characterized amnion-derived multipotent progenitor cells, or AMP cells, by in vitro methods and showed they were able to inhibit antigen-specific T-cell proliferation in a cell-to-cell contact-dependent fashion. Here we examine specific mechanisms involved in immunomodulation by AMP cells. We found that AMP cells significantly inhibited monocyte-derived myeloid dendritic cell (DC) maturation when placed in coculture. Cocultured monocytes retained the nondifferentiated macrophage marker CD14 while exhibiting significant reduction in DC maturation markers CD83 and CD1a, indicating an immature DC maturation state that is pivotal in determining its immune stimulatory or regulatory status. This effect was again dependent on cell-to-cell contact interaction. We also found a significant shift in cytokines present in the microenvironment of cocultures, which indicated a regulatory DC function rather than a stimulatory cell type. Here supernatants taken from AMP cell/monocyte cocultures yielded significant levels of regulatory cytokines, such as PGE2, IL-6, IL-10, and MIC-1. The soluble form of HLA-G was also found at higher levels in cocultures. In contrast, supernatants contained significantly less amounts of the T-cell-stimulating factor IL-12, which is normally produced by activated DCs. Interestingly, cocultured monocytes acquired significant expression of HLA-G on their cell surface over time. HLA-G has multifaceted immunological implications and may be a key molecule in influencing these cells to behave as regulatory DCs. Together, the influence of AMP cells on maturing DCs may favor a regulatory pathway that can be useful for therapeutic applications for immune-mediated disorders or transplantation therapies.

Duration and magnitude of hypotension and monocyte deactivation in patients with community-acquired pneumonia.

The objective was to examine the relationship of duration and magnitude of arterial hypotension to subsequent cellular immune suppression and cytokinemia in patients hospitalized with community-acquired pneumonia (CAP). We studied an observational cohort of 525 subjects hospitalized after presenting to the emergency department with radiographic and clinical signs of CAP. We compared the duration and magnitude of hypotension, using the cardiovascular Sequential Organ Failure Assessment (CV SOFA) subscore, to day 3 monocyte expression of human leukocyte antigen-DR (mHLA-DR), a previously validated marker of cellular immune suppression. A significant association of CV SOFA with decreased mHLA-DR expression was present in univariate analysis (P < 0.001) and persisted after adjustment for illness severity and other covariates (P = 0.01). With CV SOFA separated into components of magnitude and duration, after covariate adjustment, only duration was associated with day 3 mHLA-DR expression (P = 0.03). Levels of key proinflammatory and anti-inflammatory cytokines (interleukin 6 [IL-6], IL-10, tumor necrosis factor) increased with hypotension exposure and were also associated with mHLA-DR expression. In patients admitted with CAP, arterial hypotension over the first 3 days is associated with markers of monocyte deactivation. The duration of exposure to hypotension may be more important than the magnitude, and monocyte deactivation correlates with IL-6 and IL-10 release. These results suggest that persistent hypotension might contribute to immunosuppression following septic shock.

Identification and expansion of a unique stem cell population from adult mouse gallbladder.

UNLABELLED: The identification of resident stem cells in the mouse gallbladder is, to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The aim of this study was to isolate stem cells from an adult mouse gallbladder and determine whether they were unique, compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM(+) CD49f(hi) epithelial cell subpopulation from primary gallbladder is enriched in colony-forming cells, compared to EpCAM(+) CD49f(lo) cells. EpCAM(+) CD49f(hi) cells expressed cluster of differentiation (CD)29, CD133, and stem cell antigen-1, but were negative for lineage markers CD31, CD45, and F4/80. Using a novel feeder cell-culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM(+) CD49f(hi) cells in vitro. In a matrigel differentiation assay, EpCAM(+) CD49f(+) cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of, primary gallbladder. EpCAM(+) CD49f(+) cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in the expression of CD49f, CD49e, CD81, CD26, CD54, and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM(+) CD49f(+) gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione S-transferase, Indian hedgehog, and solute carrier family genes. CONCLUSION: We have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique, compared to IHBD cells.

A stochastic model for cancer stem cell origin in metastatic colon cancer.

Human cancers have been found to include transformed stem cells that may drive cancer progression to metastasis. Here, we report that metastatic colon cancer contains clonally derived tumor cells with all of the critical properties expected of stem cells, including self-renewal and the ability to differentiate into mature colon cells. Additionally, when injected into mice, these cells initiated tumors that closely resemble human cancer. Karyotype analyses of parental and clonally derived tumor cells expressed many consistent (clonal) along with unique chromosomal aberrations, suggesting the presence of chromosomal instability in the cancer stem cells. Thus, this new model for cancer origin and metastatic progression includes features of both the hierarchical model for cancerous stem cells and the stochastic model, driven by the observation of chromosomal instability.

Proinflammatory cytokines increase the rate of glycolysis and adenosine-5'-triphosphate turnover in cultured rat enterocytes.

OBJECTIVE: Measurements of steady-state adenosine-5'-triphosphate (ATP) levels in tissue samples from patients or experimental animals with sepsis or endotoxemia provide little information about the rate of ATP production and consumption in these conditions. Accordingly, we sought to use an in vitro "reductionist" model of sepsis to test the hypothesis that proinflammatory cytokines modulate ATP turnover rate. DESIGN: In vitro "reductionist" model of sepsis. SETTING: University laboratory. SUBJECTS: Cultured rat enterocyte-like cells. INTERVENTIONS: IEC-6 nontransformed rat enterocytes were studied under control conditions or following incubation for 24 or 48 hrs with cytomix, a mixture of tumor necrosis factor-alpha (10 ng/mL), interleukin-1beta (1 ng/mL), and interferon-gamma (1000 units/mL). To measure ATP turnover rate, ATP synthesis was acutely blocked by adding to the cells a mixture of 2-deoxyglucose (10 mM), potassium cyanide (8 mM), and antimycin A (1 microM). ATP content was measured at baseline (before metabolic inhibition) and 0.5, 1, 2, 5, and 10 mins later. Log-linear ATP decay curves were generated and the kinetics of ATP utilization thereby calculated. MEASUREMENTS AND MAIN RESULTS: ATP consumption rate was higher in cytomix-stimulated compared with control cells (3.11 +/- 1.39 vs. 1.25 +/- 0.66 nmol/min, respectively; p <.01). Similarly, the half-time for ATP disappearance was shorter in cytomix-stimulated compared with control cells (2.63 +/- 1.00 vs. 6.21 +/- 3.49; p <.05). In contrast to these findings, the rate of ATP disappearance was similar in cytokine-naïve and immunostimulated IEC-6 cells when protein and nucleic acid synthesis were inhibited by adding 50 microg/mL cycloheximide and 5 microg/mL actinomycin D to cultures for 4 hrs. The rates of glucose consumption and lactate production were significantly greater in cytomix-stimulated compared with controls cells. CONCLUSIONS: Incubation of IEC-6 cells with cytomix significantly increased ATP turnover. Increased ATP turnover rate was supported by increases in the rate of anaerobic glycolysis. These findings support the view that proinflammatory mediators impose a metabolic demand on visceral cells. In sepsis, cells may be more susceptible to dysfunction on the basis of diminished oxygen delivery and/or mitochondrial dysfunction.