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Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches

The epithelial lining of the small intestine consists of multiple cell types, including Paneth and goblet cells, that work in cohort to maintain gut health. The cells serve to mediate signals between gut microbiota and the host innate/adaptive immune system. The disruption of epithelial homeostasis along with dysregulated immune responses leads to the development of inflammatory bowel disease (IBD), including Crohn's (CD) and ulcerative colitis (UC). Previously, it has been observed that dysfunctional Paneth cells with reduced secretion of antimicrobial peptides contribute to the pathogenesis of CD, while reduction in goblet cell numbers and defective mucus production was associated with UC in humans.

To provide a deeper insight into the role of specific cell populations in a healthy gut and in IBD, in vitro organoid models have been increasingly employed as a research tool. Organoids, which can be derived from primary tissues or iPS cells, contain all the major cell types of the intestinal epithelium and are easily expanded in culture, manipulated and analysed. Recently, small molecule treatments have been developed that direct the differentiation of organoids towards Paneth cell or goblet cell populations. These compounds include inhibitors of Notch signaling and GSK3β-mediated β (for differentiation into Paneth cells linage) or inhibitors of Wnt signaling (for enrichment of organoid cultures in goblet cells). While these methods do not present single cell resolution, they provide a useful tool to study Paneth and goblet cell populations in the context of the other epithelial cell types.

The authors of this report aimed to comprehensively profile coding RNAs (mRNA), micro RNAs (miRNA) and long non-coding RNAs (lncRNA) from conventionally differentiated mouse organoids and compare them to Paneth cell-enriched (PCeO) and goblet cell enriched (GCeO) mouse organoids in order to predict key molecular regulators involved in Paneth cell and goblet cell specific functions.

The RNA sequencing data obtained from each set of organoids was analysed for differential expression levels (including protein coding genes, miRNAs and lncRNAs). 4135 genes were differentially expressed in the PCeO dataset, while only 2889 were differentially expressed in the GCeO dataset. Although the majority of differentially expressed genes were annotated as protein coding, a small proportion were identified as miRNAs and lncRNAs. The result suggests a shared differentiation history and secretory function of both Paneth and goblet cells.

The control and both Paneth or goblet cells enriched organoids expressed specific gut cell types markers including: Lgr5 (stem cells), ChgA (enteroendocrine cells), Muc2 (goblet cells), Lyz1 (Paneth cells) and Vil1 (epithelial cells), however upregulation of Muc2, Lyz1 and ChgA and downregulation of Lgr5 was also observed in PCeO and GCeO organoids. In addition, a number of Paneth cell specific antimicrobial peptide genes were upregulated in PCeO, while goblet cell mucin related genes were differentially expressed in both the enriched organoids' datasets, a higher fold of change was also observed in GCeO cultures. This confirmed that both organoid enrichment protocols were successful in increasing the proportion of their target cell type, but also in increasing of enteroendocrine secretory cells.

Pathway analysis showed that PCeO-specific differentially expressed genes were associated with a number of metabolic pathways, including the metabolism of vitamins and cofactors, and cholesterol synthesis. On the other hand, GCeO-specific differentially expressed genes were associated with the cell cycle through pathways such as cell cycle checkpoints, DNA replication and G1/S transition. The shared cohort of differentially expressed genes identified in PCeOs and GCeOs were associated with pathways linked to hormones, energy metabolism and chemical synapses transmission, suggesting that PCeO and GCeO organoids were indeed enriched in enteroendocrine cells.

The authors also attempted to reconstruct regulatory networks and establish regulator-target relationships within differentially expressed genes. Using the MCODE software, they have identified five distinct clusters of genes in the PCeO network and seven in the GCeO networks. The clusters identified within GCeO were associated with the endosomal/vacuolar pathway and antigen prestation, but also the cell cycle. While PCeO clusters were associated with nuclear receptor transcription pathway, regulation of lipid metabolism and senescence, but also endosomal/vacuolar pathway, iron metal response and G alpha signaling events.

More specifically, they predicted key regulators of cell type-specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1B and Myc specific to goblet cells and Ets1, Nr3c1 and Vdr shared between them. Five of those genes (Ets1, Nr1d1, Rxra, Nr3c1 and Vdr) have been previously associated with inflammation, autophagy and, more interestingly, with IBD.

Links identified between cell type regulators and cellular phenotypes of IBD suggested that global regulatory rewiring during and after differentiation of Paneth and goblet cells in the context of epithelium could contribute to IBD pathology. Future application of cell type enriched organoids combined with gene profiling can be used to dissect the possible regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

Reference

Treveil et al, Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches. Molecular Omics, The Royal Society of Chemistry, 2019

https://doi.org/10.1039/c9mo00130a

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Epistem offers validated small intestinal in vitro organoid models as a robust screening tool to aid the selection of candidate treatments with minimum GI toxicity and maximum efficacy in preventing or treating GI mucositis.

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