Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound‐associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE (2)) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β‐catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE (2)‐Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE (2) and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury

Long-term culture captures injury-repair cycles of colonic stem cells

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hop

A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using air-liquid interface (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion \u3e 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions

Canonical Wnt signals combined with suppressed TGFβ/BMP pathways promote renewal of the native human colonic epithelium

Background: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. Objective: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFβ/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. Design: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. Results: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFβ or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. Conclusions: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFβ/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population

Deciphering the complex signalling systems that regulate intestinal epithelial cell death processes and shedding

Intestinal epithelial cells play a fundamental role in maintaining homeostasis. Shedding of intestinal cells in a controlled manner is critical to maintenance of barrier function. Barrier function is maintained during this shedding process by a redistribution of tight junctional proteins to facilitate closure of the gap left by the shedding cell. However, despite the obvious importance of epithelial cell shedding to gut health a central question is how the extrusion of epithelial cells is achieved, enabling barrier integrity to be maintained in the healthy gut and restored during inflammation remains largely unanswered. Recent studies have provided evidence that excessive epithelial cell shedding and loss of epithelial barrier integrity is triggered by exposure to lipopolysaccharide (LPS) or tumour necrosis factor (TNF). Subsequent studies have provided evidence of the involvement of specific cellular components and signalling mechanisms as well as the functionality of microbiota that can be either detrimental or beneficial for intestinal barrier integrity. This review, will focus on the evidence and decipher how the signalling systems through which the mucosal immune system and microbiota can regulate epithelial cell shedding and how these mechanisms interact to preserve the viability of the epithelium

Notch-Regulated Mechanisms of Epithelial Cell Fate Selection in the Intestine.

Throughout the lifetime of an organism, progenitor cells in the intestine proliferate and differentiate to form cells of the secretory and absorptive lineages. Many intercellular signaling pathways, including the Notch pathway, coordinate to develop and maintain the intestine. My thesis work has investigated how Notch signaling regulates intestinal cell fate using several novel genetically engineered and pharmacological mouse models. Developmental analysis of a transgenic mouse model with forced expression of the Notch-regulated transcription factor Mouse atonal homolog 1 (Math1) showed increased numbers of all secretory cell types and loss of absorptive cells, demonstrating that Math1 is the key factor regulating intestinal secretory cell differentiation. Furthermore, these data suggest that Math1 can redirect a bipotential progenitor cell to the secretory cell fate. To study the role of Math1 in adult intestine, I describe an inducible Math1 transgenic model; however, Math1 protein was not increased subsequent to transgene activation, suggesting that there may be active degradation of Math1 protein in the intestine. Previous studies have shown that inhibition of Notch signaling resulted in decreased epithelial cell proliferation and altered cell fate, suggesting that a stem or progenitor cell is targeted by Notch signaling; however, the identity of this target was unknown. Pharmacological inhibition of Notch signaling in both fetal and adult intestine showed that expression of Olfactomedin 4 (Olfm4), a crypt base columnar stem cell gene, was markedly decreased upon Notch inhibition. Transcriptional studies in the human colon cancer cell line LS174T confirmed that Notch signaling activated OLFM4 gene expression and identified a region containing critical cis-regulatory DNA elements. Finally, I made the novel observation that a population of intermediate cells that express both goblet and Paneth cell markers emerged upon Notch inhibition in adult mouse ileum and colon. In conclusion, my thesis research has shown that Math1 is the key regulator of secretory cell differentiation in the intestine and that Notch signaling directly targets the crypt base columnar stem cell. Collectively, these studies have provided important information about Notch-regulated mechanisms of intestinal development and cell lineage determination.Ph.D.Molecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77890/1/kellivd_1.pd

EMT-Induced Stemness and Tumorigenicity Are Fueled by the EGFR/Ras Pathway

10.1371/journal.pone.0070427PLoS ONE88-POLN