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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

Conventional in-vitro models of intestinal epithelium such as human intestinal/colonic cancer cell lines lack essential features of the canine intestine, limiting their use to study canine intestinal barrier function, cellular metabolism, or host-microbe interactions. Intestinal organoids, also known as enteroids, represent a recent primary cell culture technology that has revolutionized the field of intestinal disease and physiology modeling. Enteroids are 3D structures that are composed of a single layer epithelium arranged in crypt- and villi-like domains with its apical and basal membranes facing the luminal and external sides of the enteroid, respectively. The conversion of the 3D organization of enteroids into a 2D monolayer enables accessibility to intestinal cells' apical membrane for experimental infections or drug treatments. This study aimed to develop a canine enteroid-derived monolayer model to study gut barrier structure and function. Jejunal segments were obtained from three donor dogs during a resection and anastomosis surgery and used to isolate the intestinal crypts by EDTA-based tissue dissociation. Then, the intestinal crypts were resuspended in growth factor reduced Matrigel and supplemented with the commercial IntestiCult™ Human Organoid Growth Medium to generate 3D enteroids. Canine enteroids were mechanically and enzymatically disrupted into single cells and cultured on Matrigel-coated porous membranes of transwell culture systems, reaching 100% confluency around day four. We performed immunofluorescence, histochemistry, and transmission electron microscopy (TEM) to study intestinal epithelium morphology and structure. Intestinal epithelium barrier function was analyzed by Fluorescein Isothiocyanate-Dextran 4kD (FITC-D4) intestinal permeability Assay and Trans-Epithelial Electrical Resistance (TEER) measurement. Canine enteroids and 2D monolayers showed positive staining for ZO-1, Ki67, and Periodic Acid of Schiff (PAS), indicating epithelial cell heterogeneity and polarization. A functional intestinal barrier was confirmed by FITC-D4 assay and TEER measurement. This protocol provides a canine-specific, complex, and long-term ex-vivo model to study intestinal epithelial function.

    Cover page: Generation of a canine enteroid-derived monolayer model to study gut barrier structure and function
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2021)
    Robust and reproducible protocols to efficiently reprogram adult canine cells to induced pluripotent stem cells are still elusive. Somatic cell reprogramming requires global chromatin remodeling that is finely orchestrated spatially and temporally. Histone acetylation and deacetylation are key regulators of chromatin condensation, mediated by histone acetyltransferases and histone deacetylases (HDACs), respectively. HDAC inhibitors have been used to increase histone acetylation, chromatin accessibility, and somatic cell reprogramming in human and mice cells. We hypothesized that inhibition of HDACs in canine fibroblasts would increase their reprogramming efficiency by altering the epigenomic landscape and enabling greater chromatin accessibility. We report that a combined treatment of panobinostat (LBH589) and vitamin C effectively inhibits HDAC function and increases histone acetylation in canine embryonic fibroblasts in vitro, with no significant cytotoxic effects. We further determined the effect of this treatment on global chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing. Finally, the treatment did not induce any significant increase in cellular reprogramming efficiency. Although our data demonstrate that the unique epigenetic landscape of canine cells does not make them amenable to cellular reprogramming through the proposed treatment, it provides a rationale for a targeted, canine-specific, reprogramming approach by enhancing the expression of transcription factors such as CEBP.
      Cover page: Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2021)
      Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell-based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine-specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC-seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell-fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine-specific reprogramming approaches.
        Cover page: Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2022)
        The small intestinal mucosa constitutes a physical barrier separating the gut lumen from sterile internal tissues. Junctional complexes between cells regulate transport across the barrier, preventing water loss and the entry of noxious molecules or pathogens. Inflammatory diseases in cattle disrupt this barrier; nonetheless, mechanisms of barrier disruption in cattle are poorly understood. We investigated the direct effects of three inflammatory cytokines, TNFα, IFNγ, and IL-18, on the bovine intestinal barrier utilizing intestinal organoids. Flux of fluorescein isothiocyanate (FITC)-labeled dextran was used to investigate barrier permeability. Immunocytochemistry and transmission electron microscopy were used to investigate junctional morphology, specifically tortuosity and length/width, respectively. Immunocytochemistry and flow cytometry was used to investigate cellular turnover via proliferation and apoptosis. Our study shows that 24-h cytokine treatment with TNFα or IFNγ significantly increased dextran permeability and tight junctional tortuosity, and reduced cellular proliferation. TNFα reduced the percentage of G2/M phase cells, and IFNγ treatment increased cell apoptotic rate. IL-18 did not directly induce significant changes to barrier permeability or cellular turnover. Our study concludes that the inflammatory cytokines, TNFα and IFNγ, directly induce intestinal epithelial barrier dysfunction and alter the tight junctional morphology and rate of cellular turnover in bovine intestinal epithelial cells.
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