A Coculture System for Modeling Intestinal Epithelial-Fibroblast Crosstalk.

Endometriomsis (EMs) is a complex and chronic gynecological disease characterized by distressing symptoms. Its pathogenesis remains unknown, and there is no effective treatment. Therefore, establishing patient-derived models is crucial for elucidating disease mechanisms and identifying potential therapeutic agents. We developed a coculture system combining epithelial organoids and stromal cells, enabling the study of their dynamic interactions. Using this model, we assessed the therapeutic efficacy of dienogest, a drug clinically used for treating endometriomas. The epithelial gland-like organoids and stromal cells derived from patients with endometriomas were isolated and cultured, respectively. Both of them were cocultured in matrix for partially mimicking invivo pathological features. Immunohistochemical (IHC) assay was used to identify their biomarkers. Cell viability was quantitatively assessed using the CellTiter-Glo® assay following drug treatment. We successfully cultured patient-derived epithelial gland-like organoids and stromal cells derived from patients with endometriomas, a form of endometriosis characterized by ovarian cysts. Morphological and immunohistochemical analyses confirmed high consistency with native endometriotic lesions. These models exhibited comparable expression profiles for key biological markers, including estrogen receptors (ERs), progesterone receptors (PRs), E-cadherin, CD44, Intercellular Adhesion Molecule-1 (ICAM1), Integrin Beta 3 (ITGB3), Cytokeratin 7 (CK7), Matrix Metalloproteinase 2/9 (MMP2/9), Tissue Inhibitor of Metalloproteinases 1 (TIMP1), and TIMP2. Notably, drug responsiveness varied among the patient-derived models by coculturing two types of cells, indicating potential interpatient heterogeneity in treatment outcomes. We propose that this patient-specific endometriomas model serves as a valuable platform for investigating disease mechanisms and screening drug in endometriomas. We established a novel coculture system integrating epithelial organoids and stromal cells to recapitulate the intricate cellular interactions within the endometriotic microenvironment, providing a more relevant invitro representation of the disease. Upon evaluation with dienogest, a clinically used therapeutic agent for endometriomas, the patient-derived models exhibited heterogeneous drug responses.

Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

Models of Pluripotent and Somatic Stem Cells to Study Tissue-Specific Sensitivities in Fanconi Anemia

Postnatal establishment of enteric metabolic, host-microbial and immune homeostasis is the result of precisely timed and tightly regulated developmental and adaptive processes. Here, we show that infection with the invasive enteropathogen Salmonella Typhimurium results in accelerated maturation of the neonatal epithelium with premature appearance of antimicrobial, metabolic, developmental, and regenerative features of the adult tissue. Using conditional Myd88-deficient mice, we identify the critical contribution of immune cell-derived mediators. Cytokine stimulation of neonatal intestinal epithelial stem cell organoids suggests a network of synergistic and antagonistic cytokine effects with a significant contribution of IL-22, IL-4/IL-13, TNF, and IL-6 to infection-induced enterocyte reprogramming. Our findings demonstrate that the infection-associated immune cell activation disrupts physiological postnatal tissue maturation and may thereby worsen clinical outcomes and alter the neonatal-adult transition.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.NEW & NOTEWORTHY We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells.

Epithelial and Mesenchymal Contribution to the Niche: A Safeguard for Intestinal Stem Cell Homeostasis

Organoid vs In Vivo Mouse Model: Which is Better Research Tool to Understand the Biologic Mechanisms of Intestinal Epithelium?

[Index to Morphogenesis Special Issue][1] ![Figure][2] CREDIT: KRISTIN BRANSON, WILLIAM LEMON, PHILIPP KELLER (JANELIA FARM RESEARCH CAMPUS, HHMI) As development progresses from a single fertilized egg to 2, 4, 8, 16 cells, and so on, the early apparent homogeneity soon transitions to cells displaying varied sizes and shapes. Cell adhesion and cortical tension, with their associated forces, contribute to such changes. Crowded cells are pushed and pulled, but some make their own way via cell-autonomous migration or chemotaxis. These events proceed in an amazingly precise, choreographed manner, both temporally and spatially. Distinct germ layers and ultimately the stereotypic body form result, with amazing robustness. This special issue presents exciting advances in understanding morphogenesis, or the development of body shape. Morphogenetic events are quickly coming into focus through new imaging techniques. Science 's online Review by Philipp Keller (p. [1234168][3]) looks at recent advances, with numerous figures and movies exemplifying the exciting possibilities to detail the dynamics of development as seen by light microscopy of live organisms. Morphogenesis can be viewed at short or long temporal scales to fit with fast subcellular rearrangements or slow organismal events, respectively, and at ever-expanding tissue depth. By combining imaging techniques with computational approaches and mathematical models, new facets of developmental discovery are made possible. Epithelia are characterized by layers of densely packed cells that are joined by strong adhesive contacts. These tissues provide structural integrity to developing embryos and adult organs and serve as barriers to pathogens and protection from dehydration. Despite what appears to be a set structural form, epithelial cells display active dynamics with fluid behavior. Guillot and Lecuit (p. [1185][4]) review mechanical aspects of epithelial cell junctions in cell division, cell extrusion, and cell intercalation, in which cells change shape and location to drive local tissue deformation. Stem cells are uniquely able to self-renew and differentiate. Recent work shows that when certain stem cells are placed in the right environment, they can self-organize to form organlike structures. Sato and Clevers (p. [1190][5]) review research in which single Lgr5 stem cells develop into epithelial organoids that resemble in vivo intestinal epithelium. This in vitro morphogenesis provides complex structures that can be used for stem cell and organ research as well as disease modeling and regenerative medicine. A collection of News articles on Mysteries of Development (p. [1156][6]) explores what we don't yet understand about how growing organisms control size, how symbionts help drive development, why brains lose so many cells as they mature, and how the fetal environment shapes later health. By continuing to probe the components, movements, and mechanics involved in development, a deeper understanding of the makings of organismal life is gained, with expanded opportunities to address events when things go awry. [1]: http://www.sciencemag.org/site/special/morpho/index.xhtml [2]: pending:yes [3]: http://www.sciencemag.org/content/340/6137/1234168.full [4]: /lookup/doi/10.1126/science.1235249 [5]: /lookup/doi/10.1126/science.1234852 [6]: http://www.sciencemag.org/content/340/6137/1156.full

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Ferroptosis triggered by STAT1- IRF1-ACSL4 pathway was involved in radiation-induced intestinal injury

Quiescence regulators for hematopoietic stem cell

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.

Stem Cells Marked by the R-Spondin Receptor LGR5

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.

Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium