技术资料

The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis,function,and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2?ISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics,UEA-1 chromatography,and site-directed mutagenesis were monitored to dissect the regulatory mechanism,identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and ?-1,2-fucosylation of ISCs. Oxidative stress,mitochondrial dysfunction,and cell apoptosis were impeded by fucose. Meanwhile,fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily,FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal,and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation,which may provide a fascinating strategy for treating intestinal injury disorders. View Publication

While readily achieved in cell lines,the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here,we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein. View Publication

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here,we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes,organoid cell sorting,and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis. View Publication

BACKGROUND AND AIMS The intestinal mucosa undergoes a continual process of proliferation,differentiation,and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation,how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase,the transcription factor atonal homolog 1,and intestinal cell differentiation markers lysozyme,mucin 2,and chromogranin A were determined by Western blot,quantitative real-time reverse transcription polymerase chain reaction,or immunofluorescence,and immunohistochemistry staining. RESULTS HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth,goblet,and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically,HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function. View Publication