Ex Vivo and In Vivo Noninvasive Imaging of Epidermal Growth Factor Receptor Inhibition on Colon Tumorigenesis Using Activatable Near-Infrared Fluorescent Probes

Near-infrared fluorescence (NIRF) imaging combined with enzyme-activatable NIRF probes has yielded promising results in cancer detection

Generation of renewable mouse intestinal epithelial cell monolayers and organoids for functional analyses

Abstract Background Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis. Results IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ∆F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the ApcMin/+ mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture. Conclusions These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds

Cloned, CD117 Selected Human Amniotic Fluid Stem Cells Are Capable of Modulating the Immune Response

Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of tumorigenicity may make AFS cells a superior source of stable, well characterized “off the shelf” immunomodulatory cells for a variety of immunotherapies

Folklore and Oral Traditions: a Collection of Folklore in Wayne and Holmes County, Ohio

This thesis investigates the study of folklore in a number of ways. First and foremost, it is a collection of some of the folklore of the Wayne and Holmes county, Ohio area. In addition, this thesis seeks to uncover patterns concerning the inheritance of oral folk traditions. To accomplish this goal qualitative data gathered during the fall and winter of 1993 has been translated directly from oral discourse to written text for preservation. The study draws on theories which discuss societal change, and seeks to explain alterations in or loss of folklore due to these changes. This study reveals that folklore does indeed suffer alterations due to displacement and replacement, thus justifying the importance of transferring oral traditions to written text for preservation

Generation of renewable mouse intestinal epithelial cell monolayers and organoids for functional analyses

Abstract Background Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis. Results IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ∆F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the Apc Min/+ mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture. Conclusions These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds

Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem cells

Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFPLow), activatable reserve IESC and enteroendocrine cells (Sox9-EGFPHigh), Sox9-EGFPSublow progenitors, and Sox9-EGFPNegative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFPLow IESC and Sox9-EGFPHigh cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging

Soluble factors released from AFS cells and BM-MSCs in response to activation.

<p>Amniotic fluid stem (AFS) cells or bone marrow derived mesenchymal stem cells (BM-MSCs) were activated by culture with A. total blood monocytes or B. IL-1β and the cytokines released were measured by cytokine array. Background cytokine levels were subtracted to normalize samples and include cytokines released by PBMCs cultured alone and stem cells cultured alone. Quantification of protein optical density was measured using GenePix4000 software.</p

Human AFS cells inhibit lymphocyte activation in a dose dependent manner similar to that of BM-MSCs.

<p>Immunoassays assessing lymphocyte activation were performed on two independent amniotic fluid stem cell lines (AFS1 and AFS2) or bone marrow-mesenchymal stem cell (BM-MSC) isolates. T lymphocytes were activated with phytohemaaglutinin (PHA) and cultured in 96 well plates coated with IFN-γ capture antibody in the presence of increasing amounts of stem cells from 1∶32 (4,688 stem cells cultured with 150,000 PBMCs) to 1∶2 (75,000 stem cells cultured with 150,000 PBMCs) for 24 hours. Positive control wells contained lymphocytes activated with PHA and negative control wells included unactivated lymphocytes. Lymphocyte activation was assessed by counting the number of lymphocyte clones producing IFN-γ. Activation is expressed as a percentage of the positive control wells. Both AFS lines and BM-MSC inhibited T-cell compared to the PHA activated control to an approximately equal extent, and was dependant on the number of stem cells added. Inhibition varied from about 40% at a 1∶32 ratio to 80–90% inhibition at the highest ratio of 1∶2.</p