Spatial Reconstruction of Single Enterocytes Uncovers Broad Zonation along the Intestinal Villus Axis.

The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking.

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.

Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.

The mammalian liver consists of hexagon-shaped lobules that are radially polarized by blood flow and morphogens. Key liver genes have been shown to be differentially expressed along the lobule axis, a phenomenon termed zonation, but a detailed genome-wide reconstruction of this spatial division of labour has not been achieved. Here we measure the entire transcriptome of thousands of mouse liver cells and infer their lobule coordinates on the basis of a panel of zonated landmark genes, characterized with single-molecule fluorescence in situ hybridization. Using this approach, we obtain the zonation profiles of all liver genes with high spatial resolution. We find that around 50% of liver genes are significantly zonated and uncover abundant non-monotonic profiles that peak at the mid-lobule layers. These include a spatial order of bile acid biosynthesis enzymes that matches their position in the enzymatic cascade. Our approach can facilitate the reconstruction of similar spatial genomic blueprints for other mammalian organs.

A single cell atlas of the human liver tumor microenvironment.

Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA-sequencing and spatial analysis of malignant and adjacent non-malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient-independent expression programs, and reconstruct a ligand-receptor map that highlights recurring tumor-stroma interactions. By combining transcriptomics of laser-capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non-malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions.

Endometrial stem cell transplantation in MPTP- exposed primates: an alternative cell source for treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. Cell-replacement therapies have emerged as a promising strategy to slow down or replace neuronal loss. Compared to other stem cell types, endometrium-derived stem cells (EDSCs) are an attractive source of stem cells for cellular therapies because of their ease of collection and vast differentiation potential. Here we demonstrate that endometrium-derived stem cells may be transplanted into an MPTP exposed monkey model of PD. After injection into the striatum, endometrium-derived stem cells engrafted, exhibited neuron-like morphology, expressed tyrosine hydroxylase (TH) and increased the numbers of TH positive cells on the transplanted side and dopamine metabolite concentrations in vivo. Our results suggest that endometrium-derived stem cells may provide a therapeutic benefit in the primate model of PD and may be used in stem cell based therapies.

Derivation of insulin producing cells from human endometrial stromal stem cells and use in the treatment of murine diabetes.

Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using a simple and nontransfection protocol. An in vitro protocol was developed and evaluated by assessing the expression of pan ß-cell markers, followed by confirmation of insulin secretion. PAX4, PDX1, GLUT2, and insulin, were all increased in differentiated cells compared to controls. Differentiated cells secreted insulin in a glucose responsive manner. In a murine model, differentiated cells were injected into the kidney capsules of diabetic mice and human insulin identified in serum. Within 5 weeks blood glucose levels were stabilized in animals transplanted with differentiated cells, however those treated with undifferentiated cells developed progressive hyperglycemia. Mice transplanted with control cells lost weight and developed cataracts while those receiving insulin producing cells did not. Endometrium provides an easily accessible, renewable, and immunologically identical source of stem cells with potential therapeutic applications in diabetes.

Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells.

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell's identity and its position in a tissue. We recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here we used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs' tissue coordinates. We applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, we reconstructed LEC zonation and extracted a landmark gene panel that we used to spatially map LEC scRNAseq data. Our approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

Global mRNA polarization regulates translation efficiency in the intestinal epithelium.

Asymmetric messenger RNA (mRNA) localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and importance of mRNA polarization in epithelial tissues are unclear. Here, we used single-molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated. Refeeding of fasted mice elicited a basal-to-apical shift in polarization of mRNAs encoding ribosomal proteins, which was associated with a specific boost in their translation. This led to increased protein production, required for efficient nutrient absorption. These findings reveal a posttranscriptional regulatory mechanism involving dynamic polarization of mRNA and polarized translation.

Migration of cells from experimental endometriosis to the uterine endometrium.

Endometriosis is the estrogen-dependent growth of endometrial tissue outside the uterus. Endometriosis has an effect on the eutopic endometrium; however, the nature of the cellular or molecular signal from the lesion to the uterus is unknown. Here we demonstrate that cells migrate from endometriosis to eutopic endometrium. Experimental endometriosis was established by transplanting endometrial tissue from green fluorescent protein (GFP) mice to the peritoneal cavity of DS-Red mice. Immunofluorescence (IF) identified cells from the ectopic lesions in the uterus. The eutopic endometrial cells were sorted by fluorescence activated cell sorting, and the GFP(+)/DS-Red(-) population was characterized using microarray analysis. The results of cell sorting as well as the array results were confirmed by quantitative PCR and IF. GFP(+)/DS-red(-)/Cd45(-) cells were identified in the eutopic endometrium of mice with experimental endometriois (∼1.8%) and not in controls. Global gene expression profiling of these cells showed absence of leukocyte and increased expression of pan-epithelial markers in the uterine GFP(+) cells. Moreover, GFP(+) cells showed up-regulation of Wnt7A expression and 17 other genes associated with the Wingless pathway. Several genes that are associated with epithelial-to-mesenchymal transition were also highly differentially expressed in GFP(+) cells. IF confirmed the presence of the GFP(+)/CD45(-)/Wnt7a(+)/cytokeritin(+) cells in the endometrium of endometriotic animals, and not in controls. Cells from endometriotic lesions are capable of migrating to the eutopic endometrium. The ectopic expression of Wnt7A suggests a possible mechanism by which ectopic lesions affect the eutopic endometrium and interfere with embryo implantation and fertility.