In vitroandin vivodevelopment of the human intestinal niche at single cell resolution

Abstract

SUMMARYThe human intestinal stem cell (ISC) niche supports ISC self-renewal and epithelial function, yet little is known about the development of the human ISC niche. We used single-cell mRNA sequencing (scRNA-seq) to interrogate the human intestine across 7-21 weeks of gestation. Using these data coupled with marker validation in situ, molecular identities and spatial locations were assigned to several cell populations that comprise the epithelial niche, and the cellular origins of many niche factors were determined. The major source of WNT and RSPONDIN ligands were ACTA2+ cells of the muscularis mucosa. EGF was predominantly expressed in the villus epithelium and the EGF-family member NEUREGULIN1 (NRG1) was expressed by subepithelial mesenchymal cells. Functional data from enteroid cultures showed that NRG1 improved cellular diversity, enhanced the stem cell gene signature, and increased enteroid forming efficiency, whereas EGF supported a secretory gene expression profile and stimulated rapid proliferation. This work highlights unappreciated complexities of intestinal EGF/ERBB signaling and identifies NRG1 as a stem cell niche factor.

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

“…Validation of the organ-specific EC genes and gene signatures identified by bulk RNAseq (Figure 3) was carried out using both scRNAseq and fluorescent in situ hybridization (FISH) (Figure 4). We used scRNAseq to profile human fetal lung (Miller et al, 2020), intestine (Czerwinski et al, 2020), and kidney across 7 specimens spanning 13.5-19 weeks of gestation (Figure S4A). In total, our analyses included 62,046 cells across these three organs (Figure 3A and S3A).…”

“…For cell type scoring of major cell classes (epithelium, mesenchyme, endothelial, neuronal, immune), gene sets for each class were curated in previously published work (Czerwinski et al, 2020; Miller et al, 2020) (Supplementary Table 2), and log normalized and z-transformed raw counts were summed to generate cell type scores. For visualization of these data, cell type scores were mapped onto UMAP embeddings.…”

“…The human fetal lung scRNAseq data used for our analyses have been previously published (Miller et al, 2020). The human fetal intestine (Czerwinski et al, 2020) and kidney data deposition is in progress. Code is available at: https://github.com/jason-spence-lab/Holloway2020…”

An organoid and multi-organ developmental cell atlas reveals multilineage fate specification in the human intestine

“…We next sought to quantify the similarity of tHIO epithe- lial and mesenchymal cells to primary human tissue, and determine the identity of off-target cells. We established a developing human multi-organ cell atlas by integrating single-cell transcriptomes from lung, liver, esophagus, stomach, small intestine (duodenum, jejunum, ileum), and colon (155,232 cells total) with an age distribution spanning 7 to 21 PCW (Figure 2A; Tables S1 and S2; Key Resources Table) [14,30,38]. We note that for some of the time points, we have sequenced multiple organs from the same human specimens ( Figure S3C).…”

“…There have been single-cell transcriptome surveys that illuminate cell composition and epithelial differentiation programs in mouse [23][24][25][26] and human [27] intestinal epithelial organoids derived from adult stem cells. Reference atlases are also being established from developing and adult primary human intestinal tissues [28][29][30][31] to identify molecular features underlying intestine development. Single-cell maps of gastrulation and other early stages of organ specification and development in mice can provide insight into processes that are difficult to explore in human primary tissues [32,33].…”