Comparison of expression profiles between undifferentiated and differentiated porcine IPEC-J2 cells

Background The intestinal porcine enterocyte cell line (IPEC-J2) is a well-established model to study porcine intestinal physiology. IPEC-J2 cells undergo spontaneous differentiation during culture while changes in expression patterns of differentiated IPEC-J2 remain unclear. Therefore, this study was aimed to investigate the expression profiles of IPEC-J2 cells at the transcriptional level. Differentially expressed genes (DEGs), enriched pathways and potential key genes were identified. Alkaline phosphatase (AKP) and percentages of apoptotic cells were also measured. Results Overall, a total of 988 DEGs were identified, including 704 up-regulated and 284 down-regulated genes. GO analysis revealed that epithelial cell differentiation, apoptotic signaling pathway, regulation of cytokine production and immune system process, regulation of cell death and proliferation, cell junction complexes, and kinase binding were enriched significantly. Consistently, KEGG, REACTOME, and CORUM analysis indicated that cytokine responses modulation may be involved in IPEC-J2 differentiation. Moreover, AKP activity, a recognized marker of enterocyte differentiation, was significantly increased in IPEC-J2 after 14 days of culture. Meanwhile, annexin V-FITC/PI assay demonstrated a remarkable increase in apoptotic cells after 14 days of culture. Additionally, 10 hub genes were extracted, and STAT1, AKT3, and VEGFA were speculated to play roles in IPEC-J2 differentiation. Conclusions These findings may contribute to the molecular characterization of IPEC-J2, and may progress the understanding of cellular differentiation of swine intestinal epithelium.

Single-Cell Transcriptome Sequencing and Proteomics Reveal Neonatal Ileum Dynamic Developmental Potentials

We found previously unknown neonatal ileum developmental potentials: specific increases in undifferentiated cells, unique enterocyte differentiation, and time dependent reduction in secretory cells. Specific transcriptional factors (TFs), ligand-receptor pairs, G protein-coupled receptors, transforming growth factor β, bone morphogenetic protein signaling pathways, and the gut mucosal microbiota are involved in this process.

Retrospective analysis of enhancer activity and transcriptome history

Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. Here, we utilize an inducible DCM-RNA-polymerase-subunit-b fusion protein, to label active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. We applied this DCM-time-machine (DCM-TM) technology to study intestinal homeostasis, following enterocyte differentiation back in time, revealing rapid and simultaneous activation of enhancers and nearby genes during intestinal stem cell (ISC) differentiation. We provide new insights in the absorptive-secretory lineage decision in ISC differentiation, and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes in development and differentiation.

miR-802 regulates Paneth cell function and enterocyte differentiation in the mouse small intestine

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Background/Aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

The effect of polymeric formula on enterocyte differentiation

Exclusive enteral nutrition is established as an initial therapy to induce remission in active Crohn’s disease (CD), especially in children, but the mechanisms of action of this therapy are yet to be fully defined. Intestinal alkaline phosphatase (IAP), a recognised marker of enterocyte differentiation, is implicated in the innate gut immune response to enteric pathogens. Using the Caco-2 human adenocarcinoma cell line, this study showed that the incubation of human cells with a polymeric formula (PF) resulted in a dose-dependent increase in the expression of IAP on the cell surface. While further investigation is required to determine the pathway(s) involved, this finding suggests that cell surface-associated IAP may be an aspect of the gut’s innate immune response to pathogenic bacteria that is strengthened by PF in the setting of CD.