recapitulating-suckling-to-weaning-transition-vitro-using-fetal

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

University of Amsterdam

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous  deletion by use of  mice and organoids. We examine models of irradiation and tumorigenesis, both  and  Although we observed no phenotypic changes in  heterozygous mice,  heterozygous organoid growth was markedly reduced. Irradiation of  heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to -mutant animals for adenoma studies and found that adenomagenesis in  heterozygous- heterozygous mice was reduced compared with  heterozygous controls (1.43 vs. 3.33;  < 0.01). In conclusion, epithelium-specific  heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue. Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. .

Heterozygosity of Chaperone Grp78 Reduces Intestinal Stem Cell Regeneration Potential and Protects against Adenoma Formation.

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation  postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation  Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.

Intestinal epithelial cells have a defined hierarchy with stem cells located at the bottom of the crypt and differentiated cells more at the top. Epithelial cell renewal and differentiation are strictly controlled by various regulatory signals provided by epithelial as well as surrounding cells. Although there is evidence that stromal cells contribute to the intestinal stem cell niche, their markers and the soluble signals they produce have been incompletely defined. Using a number of established stromal cell markers, we phenotypically and functionally examined fibroblast populations in the colon. CD90+ fibroblasts located in close proximity to stem cells in vivo support organoid growth in vitro and express crucial stem cell growth factors, such as Grem1, Wnt2b, and R-spondin3. Moreover, we found that CD90+ fibroblasts express a family of proteins-class 3 semaphorins (Sema3)-that are required for the supportive effect of CD90+ fibroblasts on organoid growth.

Colonic CD90+ Crypt Fibroblasts Secrete Semaphorins to Support Epithelial Growth.

Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.

The P4-ATPase ATP9A is a novel determinant of exosome release.

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF6. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

Article	Year
Heterozygosity of Chaperone Grp78 Reduces Intestinal Stem Cell Regeneration Potential and Protects against Adenoma Formation. Cancer research\| PubMed ID: 30232220	2018
Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning. EMBO reports\| PubMed ID: 30530633	2019
Colonic CD90+ Crypt Fibroblasts Secrete Semaphorins to Support Epithelial Growth. Cell reports\| PubMed ID: 30917322	2019
The P4-ATPase ATP9A is a novel determinant of exosome release. PloS one\| PubMed ID: 30947313	2019
Expression of UPR effector proteins ATF6 and XBP1 reduce colorectal cancer cell proliferation and stemness by activating PERK signaling. Cell death & disease\| PubMed ID: 31227689	2019

Vanesa Muncan

Biography

JoVE Journal Publications

Other Publications