Author Correction: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Change history: In this Letter, the surname of author Efi E. Massasa was misspelled 'Massassa'. This error has been corrected online.

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL-6 trans-signaling.

BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.

Telocytes in the Luminal GI Tract.

Telocytes are unique mesenchymal cells characterized by multiple remarkably long cytoplasmic extensions that extend hundreds of micron away from the cell body. Through these extensions, telocytes establish a 3-dimensional network by connecting with other telocytes and various cell types within the tissue. In the intestine, telocytes have emerged as an essential component of the stem cell niche, providing Wnt proteins that are critical for the proliferation of stem and progenitor cells. However, the analysis of single-cell RNA sequencing has revealed other stromal populations and mechanisms for niche organization, raising questions about the role of telocytes as a component of the stem cell niche. This review explores the current state-of-the-art, existing controversies, and potential future directions related to telocytes in the luminal gastrointestinal tract.

Telocytes: Detection, Visualization, Tissue Dissociation, and Tamoxifen-Induction of Transgenic Mice.

Telocytes, distinctive interstitial cells, have recently emerged as crucial components of the stem-cell niche in the intestine. Notably, telocytes are distinguished by their extremely long cellular protrusions extending hundreds of microns from the cell body, forming an interconnected network along the intestinal crypt villus axis. Due to these unique cellular characteristics, there is a need for tailored working protocols to effectively characterize and target telocytes. Here, we outline advanced and progressive protocols for tissue fixation, dissociation, visualization, and the use of tamoxifen-induced transgenic mouse models to specifically target telocytes.

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation. Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes. The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

TET2 and TET3 loss disrupts small intestine differentiation and homeostasis.

TET2/3 play a well-known role in epigenetic regulation and mouse development. However, their function in cellular differentiation and tissue homeostasis remains poorly understood. Here we show that ablation of TET2/3 in intestinal epithelial cells results in a murine phenotype characterized by a severe homeostasis imbalance in the small intestine. Tet2/3-deleted mice show a pronounced loss of mature Paneth cells as well as fewer Tuft and more Enteroendocrine cells. Further results show major changes in DNA methylation at putative enhancers, which are associated with cell fate-determining transcription factors and functional effector genes. Notably, pharmacological inhibition of DNA methylation partially rescues the methylation and cellular defects. TET2/3 loss also alters the microbiome, predisposing the intestine to inflammation under homeostatic conditions and acute inflammation-induced death. Together, our results uncover previously unrecognized critical roles for DNA demethylation, possibly occurring subsequently to chromatin opening during intestinal development, culminating in the establishment of normal intestinal crypts.

Lineage Tracing of FOXL1+ Cells in the Tunica Muscularis Suggests Mutual Origin for Telocytes and Smooth Muscle Cells.

We recently identified a FOXL1+ intestinal subepithelial network of telocytes (TCs) without which epithelial stem and progenitor cells cannot proliferate and support regeneration. In addition to FOXL1 lineage cell distribution along the intestinal epithelium, we also observed their presence within the muscle layers. Here, we characterized FOXL1+ lineage cells along the muscle layers of the duodenum in order to understand their progeny and relation to interstitial Cajal cells (ICCs), smooth muscle cells (SMCs) and the previously reported PDGFRa+ TCs. Using a FOXL1-Cre transgenic line in conjunction with genetic lineage labeling using the Rosa26-mTmG allele, in which Cre-marked cells produce a membrane-targeted version of green fluorescent protein (GFP), we found that within the muscle layers FOXL1 lineage GFP+ cells had two main progeny; (i) elongated multinucleated SMA+ SMCs, intermingled in parallel or perpendicular to muscle fibers. (ii) TCs displaying small cell body with multiple cell processes, expressing PDGFRa and CD34. These findings may suggest a mutual origin for TCs and SMCs.

Lgr5+ telocytes are a signaling source at the intestinal villus tip.

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.

Foxl1-expressing mesenchymal cells constitute the intestinal stem cell niche.

BACKGROUND & AIMS: Intestinal epithelial stem cells that express Lgr5 and/or Bmi1 continuously replicate and generate differentiated cells throughout life1. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells2. However, ablating Paneth cells has no effect on maintenance of functional stem cells3-5. Here, we demonstrate definitively that a small subset of mesenchymal, subepithelial cells expressing the winged-helix transcription factor Foxl1 are a critical component of the intestinal stem cell niche. METHODS: We genetically ablated Foxl1+ mesenchymal cells in adult mice using two separate models by expressing either the human or simian diphtheria toxin receptor (DTR) under Foxl1 promoter control. CONCLUSIONS: Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor-cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells.