Mapping Development of the Human Intestinal Niche at Single-Cell Resolution

Abstract

The human intestinal stem cell niche supports self-renewal and epithelial function, but little is known about its development. We used single-cell mRNA sequencing with in situ validation approaches to interrogate human intestinal development from 7-21 weeks post conception, assigning molecular identities and spatial locations to cells and factors that comprise the niche. Smooth muscle cells of the muscularis mucosa, in close proximity to proliferative crypts, are a source of WNT and RSPONDIN ligands, whereas EGF is expressed far from crypts in the villus epithelium. Instead, an PDGFRA^HI/F3^HI/DLL1^HI mesenchymal population lines the crypt-villus axis and is the source of the epidermal growth factor (EGF) family member NEUREGULIN1 (NRG1). In developing intestine enteroid cultures, NRG1, but not EGF, permitted increased cellular diversity via differentiation of secretory lineages. This work highlights the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine.

Keywords: NRG1; development; enteroid; human; intestine; niche; organoid; scRNA-seq; stem cell.

Figure 1.. Mesenchymal heterogeneity in the developing human duodenum.

(A) H&E staining on human fetal intestine sections at a constant scale spanning from 54d to 130d (days post conception) (B) Timeline of specimens and corresponding number of cells profiled by scRNA-seq after filtering and ‘cleaning’ of ambient/background RNA (see STAR Methods) (n=8 biological specimens, n=1 technical replicate 47d, 59d, 72d, 80d, 101d, and 132d, n=2 technical replicates 122d and 127d). (C) UMAP visualization of each sample analyzed by scRNA-seq displayed by the age post conception. Clusters identities were assigned based on expression of canonical lineage markers (see also Figure S1). Mesenchymal (green), epithelial (blue), neuronal (yellow), immune (purple), and endothelial (pink) cell clusters were identified in all ages sequenced. (D) Following application of Harmony to mesenchymal cells from all time points, a force directed layout illustrates the relationship between timepoints. Cells are colored by sample identity (days post conception). (E) Feature plots of individual genes for various lineages are shown, including PDGFRA, F3, DLL1, NPY, GPX3, TAGLN, ANO1, and RGS5 plotted onto the force-directed layout presented in Figure 1C. (F) Representative images from FISH staining for F3 (pink) and immunofluorescent protein staining for SM22 (TAGLN protein product) with DAPI (grey) on the developing human intestine (n=1 biological replicate per timepoint) Scalebars represent 200μm. (G) Spatial characterization of PDGFRA^HI/DLL1^HI/F3^HI and GPX3^HI mesenchymal cells using FISH in the developing human intestine. Multiplexed FISH/IF for F3, PDGFRa, DLL1, NPY, GPX3 (pink or green) counterstained with DAPI (grey) and in one case ECAD (blue). Representative data is shown for n=1 biological replicate aged 132d for DLL1/F3, NPY/F3, and NPY/GPX3 and n=2 biological replicates for PDGFRa/F3, 120d specimen is shown. Scalebars represent 25 μm.

Figure 2.. Interrogating stem cell niche factors in the developing human intestine.

(A) Summary schematic annotating the approximate expression domains of several mesenchymal subpopulations on the force directed layout as identified in Figure 1E and Figure S1C. (B) Feature plots of several individual ISC niche factors including EGF, NRG1, WNT2B, RSPO2, and RSPO3 in mesenchymal cells at all time points profiled. (C) Multiplexed FISH for niche factors EGF (green), NRG1 (green), WNT2B (pink), RSPO2 (pink), and RSPO3 (pink) coupled with immunofluorescent protein staining of SM22 (blue), DAPI (grey), and FISH for F3 (green) in developing human fetal crypts. Representative data are shown from n=2 biological replicates, 140d specimen shown for EGF and NRG1, while 132d specimen shown for RSPO2, RPSO3, and WNT2B. Lower magnification images for all panels are presented in Figure S2. (D) Following application of Harmony to epithelial cells from all time points, force directed layout illustrates the relationship among timepoints. Cells are colored by sample identity (days post conception). (E) Feature plots of EGFR, ERBB2, ERBB3, and ERBB4 plotted onto the force directed layout presented in Figure 2D. (F) FISH staining in developing human fetal crypts for EGFR (pink), ERBB2 (green), and ERBB3 (red) coupled with immunofluorescent staining for ECAD (blue) and DAPI (grey) Representative data are shown from n=1 132d biological specimen. Lower magnification images for all panels are presented in Figure S2. (G) Feature plots of EGF and the enterocyte marker FABP2 plotted onto the force-directed layout presented in Figure 2D. (H) Representative images of multiplexed FISH for EGF (pink) and NRG1 (green), coupled with immunofluorescent protein staining of MKI67 (blue) and DAPI (grey) (n=1 132d human fetal intestine). The white dotted line in images roughly defines the epithelial-mesenchymal boundary. Scalebars represent 25 μm.

Figure 3.. NRG1 does not support proliferation and growth of established enteroids lines

(A) Experimental schematic for data presented in 3A. (B) Representative stereomicroscope images after 5 days of growth in the presence of EGF (100ng/ml) or NRG1 (100ng/ml). Scalebars represent 500μm. Representative data shown from n = 2 biological replicates, 142d fetal sample shown(C) Representative images of FISH staining for OLFM4 (pink) or immunofluorescent protein staining for MKI67 (pink) coupled with ECAD (blue) and DAPI (grey) in enteroids grown in EGF (100ng/ml) or NRG1 (100ng/ml). Representative data shown from n = 2 biological replicates, 142d fetal sample shown. Scalebars represent 100μm. (D) UMAP embedding of enteroid scRNAseq data (5,509 cells total) demonstrating the 5 precited clusters (n = 1 biological sample sequenced, 142d fetal sample). (E) UMAP embedding of enteroid scRNAseq data colored by culture condition (EGF- 2,789 cells; NRG1– 2,720 cells). (F) Feature plot of MKI67 illustrating that most proliferating cells are within cluster 4. (F) Bar chart depicting the percentage of cells in cluster 4 from each treatment group. (H) Feature plots demonstrating the expression of the stem cell marker OLFM4, secretory progenitor marker SPDEF, and enterocyte marker FABP1. (I) Experimental schematic for enteroid forming assays (left). Stereoscope images of enteroids after single-cell passaging and 10 day growth without EGF-family ligands (control) or in the presence of EGF (100ng/ml), NRG1 (100ng/ml), or both EGF and NRG1 (100ng/ml each). (representative data shown from n = 2 biological replicates, 142d fetal sample shown) Scalebars represent 500μm.

Figure 4.. Establishment of new enteroid lines in NRG1 increases cell type diversity in vitro.

(A) Experimental schematic. Enteroids were established from 132d (B-C) or 105d (D-J) human fetal specimen in the presence of EGF (100ng/ml), NRG1 (100ng/ml), both EGF (100ng/ml) and NRG1 (100ng/ml), or without any EGF or NRG1. Representative data shown from n = 4 biological replicates in total – 74d, 101d, 105d, 132d. (B) Representative stereoscope images of enteroids in each condition 8 days after placing isolated epithelium in Matrigel with growth factors. Scalebars represent 1mm (C) Representative images of FISH for OLFM4 (pink) or immunofluorescent protein staining for MKI67 (pink), MUC2 (pink), LYZ (pink) and ECAD (blue) and DAPI (grey) in enteroids after P0, 11 days of growth in the presence of EGF, NRG1, or both EGF and NRG1. Scalebars represent: 100μm. (D-J) scRNA-seq was performed on enteroids from EGF, NRG1, and dual EGF/NRG1 conditions after P1, 11 days of in vitro growth (n = 1 biological sample sequenced, 105d fetal sample). (D) UMAP embeddings of 13,205 enteroid cells colored by cluster identity. (E) UMAP embeddings of enteroid cells colored by sample identity (EGF- 3,262 cells, NRG1– 7,350 cells, and EGF/NRG1– 2,593 cells). (F) Bar charts depicting the cell type abundance (% of cells total sequenced) for each condition. Colors in graph correspond to Figure 4D. (G) Dotplots for the proliferation markers MKI67 and TOP2A. Both markers were enriched in clusters 4 and 8. (H) Bar chart depicting the proportion of cells sequenced that map to proliferative clusters (cluster 4 or 8) in each condition. (I) Feature plots for intestinal epithelial lineages include ISCs (OLFM4, clusters 0, 1, 2, 3, 4, 8), enterocytes (FABP2, SI - cluster 6), enteroendocrine cells (CHGA - cluster 12), and goblet cells (MUC2 - cluster 11). LYZ was broadly expressed across all enteroids conditions. (J) Bar chart depicting the proportion of cells sequenced for each condition are present in cluster 6 (enterocytes), cluster 12 (enteroendocrine cells), and cluster 11 (goblet cells). (K) Epithelial cells (828 cells) from primary intestine specimens (n= 4; 101d, 122d, 127d, 132d) were computationally extracted, re-clustered, and visualized using UMAP. (L) Cluster identities were assigned based on expression of canonical lineage markers (see Table S3 for differentially expressed genes in each cluster). (M) The Ingest function was used to map enteroids derived in the presence of EGF (100ng/ml), NRG1 (100ng/ml), or both EGF and NRG1 (100ng/ml) onto to primary intestinal epithelium reference presented in 4K. (N) The abundance of cells mapping to each of the 9 clusters identified in the in vivo intestinal epithelium was determined for the primary intestinal epithelium and for enteroids in each treatment group. Colors in graph correspond to Figure 4K.