Cells of the human intestinal tract mapped across space and time

Acknowledgements We acknowledge support from the Wellcome Sanger Cytometry Core Facility, Cellular Genetics Informatics team, Cellular Generation and Phenotyping (CGaP) and Core DNA Pipelines. This work was financially supported by the Wellcome Trust (W1T20694, S.A.T.; 203151/Z/16/Z, R. A. Barker.); the European Research Council (646794, ThDefine, S.A.T.); an MRC New Investigator Research Grant (MR/T001917/1, M.Z.); and a project grant from the Great Ormond Street Hospital Children’s Charity, Sparks (V4519, M.Z.). The human embryonic and fetal material was provided by the Joint MRC/Wellcome (MR/R006237/1) Human Developmental Biology Resource (https://www.hdbr.org/). K.R.J. holds a Non-Stipendiary Junior Research Fellowship from Christ’s College, University of Cambridge. M.R.C. is supported by a Medical Research Council Human Cell Atlas Research Grant (MR/S035842/1) and a Wellcome Trust Investigator Award (220268/Z/20/Z). H.W.K. is funded by a Sir Henry Wellcome Fellowship (213555/Z/18/Z). A.F. is funded by a Wellcome PhD Studentship (102163/B/13/Z). K.T.M. is funded by an award from the Chan Zuckerberg Initiative. H.H.U. is supported by the Oxford Biomedical Research Centre (BRC) and the The Leona M. and Harry B. Helmsley Charitable Trust. We thank A. Chakravarti and S. Chatterjee for their contribution to the analysis of the enteric nervous system. We also thank R. Lindeboom and C. Talavera-Lopez for support with epithelium and Visium analysis, respectively; C. Tudor, T. Li and O. Tarkowska for image processing and infrastructure support; A. Wilbrey-Clark and T. Porter for support with Visium library preparation; A. Ross and J. Park for access to and handling of fetal tissue; A. Hunter for assistance in protocol development; D. Fitzpatrick for discussion on developmental intestinal disorders; and J. Eliasova for the graphical images. We thank the tissue donors and their families, and the Cambridge Biorepository for Translational Medicine and Human Developmental Biology Resource, for access to human tissue. This publication is part of the Human Cell Atlas: https://www.humancellatlas.org/publications.Peer reviewedPublisher PD

Cells of the human intestinal tract mapped across space and time.

Funder: Medical Research CouncilThe cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors.

Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors

Embryonic cerebrospinal fluid nanovesicles carry evolutionarily conserved molecules and promote neural stem cell amplification.

During brain development, neural stem cells (NSCs) receive on-or-off signals important for regulating their amplification and reaching adequate neuron density. However, how a coordinated regulation of intracellular pathways and genetic programs is achieved has remained elusive. Here, we found that the embryonic (e) CSF contains 10¹² nanoparticles/ml (77 nm diameter), some of which were identified as exosome nanovesicles that contain evolutionarily conserved molecules important for coordinating intracellular pathways. eCSF nanovesicles collected from rodent and human embryos encapsulate protein and microRNA components of the insulin-like growth factor (IGF) signaling pathway. Supplementation of eCSF nanovesicles to a mixed culture containing eNSCs activated the IGF-mammalian target of rapamycin complex 1 (mTORC1) pathway in eNSCs and expanded the pool of proliferative eNSCs. These data show that the eCSF serves as a medium for the distribution of nanovesicles, including exosomes, and the coordinated transfer of evolutionary conserved molecules that regulate eNSC amplification during corticogenesis

Rodent eCSF nanovesicle purification and protein expression.

<p>(<b>A</b>) Flow chart of the experimental design after CSF labeling with a tracer dye (fast green) and eCSF collection. (<b>B</b>) Histogram of size distribution of eCSF nanoparticles (per ml) determined by nanoparticle tracking analysis (NanoSight). The nanoparticles were obtained from e14 CSF from three rat litters (N = 3). The mean nanoparticle diameter was 77 nm and was obtained by Gaussian curve fitting. (<b>C</b>) Electron micrographs of rat embryonic purified nanovesicles. Scale bar: 30 nm. (<b>D</b>) Immunoblots for rat exosomal marker proteins CD63 and HSP70, and additional proteins known to be in exosomes. PTEN and PKM2. (<b>E</b>) Quantification of phosphoenol pyruvate kinase enzymatic activity determined from rat nanovesicles. (<b>F</b>) Quantification of IGF pathway-related proteins in nanovesicles isolated from e15 rat CSF using the phospho (p)-pathscan assay. Error bars: SEM. Experiments were reproduced with nanovesicles isolated from three litters (N = 3).</p

Nanovesicles from eCSF increase IGF-mTORC1 activity in eNSCs <i>in vitro</i>.

<p>(<b>A</b>) Control and nanovesicle-treated eNSCs <i>in vitro</i> immunostained for phospho-(p) S6 (red), and nestin (green), and counterstained for the nuclear marker DAPI (blue). (<b>B</b>) Zoom of the image in the white square in (A). (<b>C</b>) Number of phospho-S6-positive cells relative to total with or without nanovesicle application (N = 3 and 4 cultures, 2–4 litters for nanovesicle extraction). Experiments were reproduced in the presence of vehicle (DMSO) or 100 nM rapamycin (N = 3 each). (<b>D</b>) Relative total cell number (N = 3 each). (<b>E</b>) Percentage of nestin-positive eNSCs (N = 3 each). (<b>F</b>) Percentage of Ki67- and nestin-positive eNSCs (N = 9 control and 3 with nanovesicle). *: p<0.05, **: p<0.01, ***: p<0.001 with Student's t test or one way ANOVA. Scale bars: 200 µm (A and B). Error bars: SEM.</p

Analysis of human eCSF nanovesicles.

<p>(<b>A</b>) Western blots for CD63, CD81, and HSP70 on purified human eCSF nanovesicles. (<b>B</b>) Pathscan analysis of human eCSF nanovesicles. All except CC3 are statistically above background at p<0.05. n.s., not significant. (<b>C</b>) Rank expression of human microRNAs based on microarray expression levels. (<b>D</b>) Differentially expressed microRNAs identified by Significant Analysis of Microarray (SAM) are indicated and represented in the heat map for human nanovesicles (left) and rat nanovesicles (right). Expression values range from low (bright green) to intermediate (black) to high (red). (<b>E</b>) Bioinformatic analysis of microRNA interacting pathways identified in eCSF purified human nanovesicles. Red microRNAs: 16-fold enriched (not shared with rat microRNAs); pink microRNA: 16-fold enriched and shared with rat; black microRNAs: 4-fold-enriched in humans and 16-fold enriched in rats. N = 4 humans. Error bars: SEM.</p

microRNA analysis of rat eCSF nanovesicles.

<p>(<b>A</b>) Heat map of microRNA microarrays from eCSF purified nanovesicles. The lighter the color (yellow) indicates higher expression whereas the darker the color (dark purple) is an indication of absence of expression. Values range from (log₂) 0.64 (bottom) to (log₂) 15.4 (top). The top 24 enriched microRNAs are listed on the right. (<b>B</b>) Rank expression of microRNAs based on microarray expression levels. (<b>C</b>) Quantitative (q) RT-PCR of exosomal RNA using selective exosomal microRNA primers or lacking primers (Neg CTL: negative control). <u>Bottom</u>: Corresponding end-point RT-PCR. (<b>D</b>) Bioinformatic analysis of microRNA interacting pathways. N = 4 litters of rats.</p