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

•Cell diversity in the developing human intestine was interrogated using scRNA-seq•PGDFRAHI/F3HI/DLL1HI mesenchyme lines the epithelium and expresses NRG1•EGF, a common in vitro niche factor, is not abundant in the crypt domain•Compared with EGF, NRG1 increases cellular diversity in enteroid culture 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 PDGFRAHI/F3HI/DLL1HI 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. 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 PDGFRAHI/F3HI/DLL1HI 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. The stem cell niche within a tissue is required to regulate stem cell maintenance, self-renewal, and differentiation (Scadden, 2006Scadden D.T. The stem-cell niche as an entity of action.Nature. 2006; 441: 1075-1079Crossref PubMed Scopus (1469) Google Scholar). The niche is made up of physical and chemical cues, including the extracellular matrix (ECM), cell-cell contacts, growth factors, and other small molecules, such as metabolites (Capeling et al., 2019Capeling M.M. Czerwinski M. Huang S. Tsai Y.-H. Wu A. Nagy M.S. Juliar B. Sundaram N. Song Y. Han W.M. et al.Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids.Stem Cell Reports. 2019; 12: 381-394Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Cruz-Acuña et al., 2017Cruz-Acuña R. Quirós M. Farkas A.E. Dedhia P.H. Huang S. Siuda D. García-Hernández V. Miller A.J. Spence J.R. Nusrat A. García A.J. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair.Nat. Cell Biol. 2017; 19: 1326-1335Crossref PubMed Scopus (270) Google Scholar; Gjorevski et al., 2016Gjorevski N. Sachs N. Manfrin A. Giger S. Bragina M.E. Ordóñez-Morán P. Clevers H. Lutolf M.P. Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (709) Google Scholar). Understanding the niche within various tissues has been central to understanding how tissues maintain homeostasis and how disease may occur (van de Wetering et al., 2002van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. et al.The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1705) Google Scholar). Further, establishing proper in vitro niche conditions has allowed growth and expansion of gastrointestinal tissue-derived stem cells in culture (Dedhia et al., 2016Dedhia P.H. Bertaux-Skeirik N. Zavros Y. Spence J.R. Organoid Models of Human Gastrointestinal Development and Disease.Gastroenterology. 2016; 150: 1098-1112Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar; Kretzschmar and Clevers, 2016Kretzschmar K. Clevers H. Organoids: Modeling Development and the Stem Cell Niche in a Dish.Dev. Cell. 2016; 38: 590-600Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). For example, by understanding that WNT signaling is important for maintaining intestinal stem cell (ISC) homeostasis (Muncan et al., 2006Muncan V. Sansom O.J. Tertoolen L. Phesse T.J. Begthel H. Sancho E. Cole A.M. Gregorieff A. de Alboran I.M. Clevers H. Clarke A.R. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc.Mol. Cell. Biol. 2006; 26: 8418-8426Crossref PubMed Scopus (202) Google Scholar; Pinto et al., 2003Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (782) Google Scholar; Sansom et al., 2004Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. et al.Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (643) Google Scholar), that blockade of Bone Morphogenetic Protein (BMP) signaling by NOGGIN (NOG) promotes ectopic crypt formation (Haramis et al., 2004Haramis A.P.G. Begthel H. Van Den Born M. Van Es J. Jonkheer S. Offerhaus G.J.A. Clevers H. De Novo Crypt Formation and Juvenile Polyposis on BMP Inhibition in Mouse Intestine.Science. 2004; 303: 1684-1686Crossref PubMed Scopus (592) Google Scholar), and that Epidermal Growth Factor (EGF) is a potent stimulator of proliferation (Goodlad et al., 1987Goodlad R.A. Wilson T.J. Lenton W. Gregory H. McCullagh K.G. Wright N.A. Proliferative effects of urogastrone-EGF on the intestinal epithelium.Gut. 1987; 28: 37-43Crossref PubMed Scopus (57) Google Scholar; Ulshen et al., 1986Ulshen M.H. Lyn-Cook L.E. Raasch R.H. Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats.Gastroenterology. 1986; 91: 1134-1140Abstract Full Text PDF PubMed Scopus (141) Google Scholar), it was determined that WNTs, RSPONDINs (RSPOs), NOG, and EGF can be utilized to expand and maintain ISCs in culture as three-dimensional intestinal organoids (Ootani et al., 2009Ootani A. Li X. Sangiorgi E. Ho Q.T. Ueno H. Toda S. Sugihara H. Fujimoto K. Weissman I.L. Capecchi M.R. Kuo C.J. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat. Med. 2009; 15: 701-706Crossref PubMed Scopus (584) Google Scholar; Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (3852) Google Scholar, Sato et al., 2011bSato T. Stange D.E. Ferrante M. Vries R.G.J. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1939) Google Scholar). This information has been leveraged to expand and culture human pluripotent stem cell-derived intestinal organoids in vitro (Capeling et al., 2020Capeling M. Huang S. Mulero-Russe A. Cieza R. Tsai Y.H. Garcia A. Hill D.R. Generation of small intestinal organoids for experimental intestinal physiology.Methods Cell Biol. 2020; 159: 143-174Crossref PubMed Scopus (9) Google Scholar; Finkbeiner et al., 2015Finkbeiner S.R. Hill D.R. 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Ongoing efforts are aimed at improving the in vitro physical environment by developing biomimetic ECM (Capeling et al., 2019Capeling M.M. Czerwinski M. Huang S. Tsai Y.-H. Wu A. Nagy M.S. Juliar B. Sundaram N. Song Y. Han W.M. et al.Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids.Stem Cell Reports. 2019; 12: 381-394Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Cruz-Acuña et al., 2017Cruz-Acuña R. Quirós M. Farkas A.E. Dedhia P.H. Huang S. Siuda D. García-Hernández V. Miller A.J. Spence J.R. Nusrat A. García A.J. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair.Nat. Cell Biol. 2017; 19: 1326-1335Crossref PubMed Scopus (270) Google Scholar; Gjorevski et al., 2016Gjorevski N. Sachs N. Manfrin A. Giger S. Bragina M.E. Ordóñez-Morán P. Clevers H. Lutolf M.P. Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (709) Google Scholar) and by adjusting signaling cues to more accurately reflect the in vivo niche (Fujii et al., 2018Fujii M. Matano M. Toshimitsu K. Takano A. Mikami Y. Nishikori S. Sugimoto S. Sato T. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition.Cell Stem Cell. 2018; 23: 787-793.e6Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). More recently, single-cell technologies have started to reveal unprecedented amounts of information about the cellular heterogeneity of human intestinal tissue and the ISC niche during health and disease (Kinchen et al., 2018Kinchen J. Chen H.H. Parikh K. Antanaviciute A. Jagielowicz M. Fawkner-Corbett D. Ashley N. Cubitt L. Mellado-Gomez E. Attar M. et al.Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.Cell. 2018; 175: 372-386.e17Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar; Martin et al., 2019Martin J.C. Chang C. Boschetti G. Ungaro R. Giri M. Grout J.A. Gettler K. Chuang L.S. Nayar S. Greenstein A.J. et al.Single-Cell Analysis of Crohn’s Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.Cell. 2019; 178: 1493-1508.e20Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar; Smillie et al., 2019Smillie C.S. Biton M. Ordovas-Montanes J. Sullivan K.M. Burgin G. Graham D.B. Herbst R.H. Rogel N. Slyper M. Waldman J. et al.Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.Cell. 2019; 178: 714-730.e22Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) and will undoubtedly yield substantial information about cell types and niche cues that regulate ISCs in various contexts. Here we set out to better understand the cellular diversity and niche cues of the developing human intestine by using single-cell mRNA sequencing (scRNA-seq) to describe the cell-associated transcriptional signatures and by using fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to define the location of cells and factors that make up the ISC niche. We sought to interrogate the cellular source of known stem cell niche factors and identify new niche factors. We determined that a source of WNT and RSPO ligands that reside just below the proliferative crypt domains is ACTA2+/TAGLN+ smooth muscle cells of the muscularis mucosa. We further determined that EGF is not expressed in the mesenchyme but is most abundant in the enterocytes of the villus epithelium, several cell diameters away from the proliferative region of the crypt. We identified a population of subepithelial cells (SECs) that lines the entire villus-crypt axis, marked by a F3HI/PDGFRAHI/DLL1HI expression profile, and found that these cells express the EGF-family ligand NEUREGULIN1 (NRG1), which has been implicated recently as an important regenerative cue in the murine intestine (Jardé et al., 2020Jardé T. Chan W.H. Rossello F.J. Kaur Kahlon T. Theocharous M. Kurian Arackal T. Flores T. Giraud M. Richards E. Chan E. et al.Mesenchymal Niche-Derived Neuregulin-1 Drives Intestinal Stem Cell Proliferation and Regeneration of Damaged Epithelium.Cell Stem Cell. 2020; 27: 646-662.e7Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Given that NRG1 is expressed in mesenchymal cells adjacent to proliferative crypts, we tested the effect of EGF and NRG1 on in vitro ISC growth using fetal human duodenal enteroids. We observed that EGF potently stimulated proliferation and primarily maintained enteroids as a stem/progenitor cell population, whereas NRG1 supported enteroid growth and enhanced cell type diversity. These results suggest that NRG1 acts as a niche cue that, when used in place of EGF, supports homeostasis in vitro, allowing stem cell maintenance and differentiation of human intestinal enteroids. Given that little is known about mesenchymal cell heterogeneity in the fetal human intestine, we aimed to better understand the mesenchymal cell populations that make up the developing human ISC niche. To do this, we obtained samples of human fetal duodenum or duodenum with adjacent proximal small intestine (in the case of very young tissues) starting just after onset of villus morphogenesis (47 days post conception) with samples interspersed up to the midpoint (132 days) of typical full-term (280 days) and performed histological and molecular analysis (Figures 1A and 1B ). Major physical changes occur throughout this developmental window: rapid growth in length and girth along with formation of villi and crypt domains within the epithelium and increased organization and differentiation of smooth muscle layers within the mesenchyme (Chin et al., 2017Chin A.M. Hill D.R. Aurora M. Spence J.R. Morphogenesis and maturation of the embryonic and postnatal intestine.Semin. Cell Dev. Biol. 2017; 66: 81-93Crossref PubMed Scopus (93) Google Scholar; Figure 1A). To capture the full complement of cell types that contribute to the developing human intestine, we dissociated full-thickness intestinal tissue from 8 specimens ranging between 47 and 132 days after conception and used scRNA-seq to sequence 2,830–3,197 cells per specimen after filtering and ambient RNA removal (STAR Methods). 24,783 total cells were used in the analysis after passing computational quality filtering (Figure 1B). Following dimensional reduction and visualization with uniform manifold approximation and projection (UMAP) (Becht et al., 2018Becht E. McInnes L. Healy J. Dutertre C.A. Kwok I.W.H. Ng L.G. Ginhoux F. Newell E.W. Dimensionality reduction for visualizing single-cell data using UMAP.Nat. Biotechnol. 2018; 37: 38-47Crossref Scopus (1311) Google Scholar; Wolf et al., 2018Wolf F.A. Angerer P. Theis F.J. SCANPY: large-scale single-cell gene expression data analysis.Genome Biol. 2018; 19: 15Crossref PubMed Scopus (1154) Google Scholar), we used canonical genes to annotate each sample individually by identifying major cell classes, including epithelial, mesenchymal, endothelial, enteric nervous, and immune cells (Figures 1C and S1A). To focus our analysis on the mesenchymal niche populations found in each sample, we computationally extracted and re-clustered the mesenchyme (1,462–2,054 cells per specimen) and annotated a population of PDGFRAHI cells, which have also been described in mice (McCarthy et al., 2020McCarthy N. Manieri E. Storm E.E. Saadatpour A. Luoma A.M. Kapoor V.N. Madha S. Gaynor L.T. Cox C. Keerthivasan S. et al.Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient.Cell Stem Cell. 2020; 26: 391-402.e5Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and ACTA2+/TAGLN+ smooth muscle cells (Figures 1E and S1C). We also identified additional sub-clusters and gene expression patterns not described previously in mice, which we describe in greater detail below (Figure S1C). Given the dramatic morphological changes that take place across this development time (Figure 1A), we implemented Harmony (Nowotschin et al., 2019Nowotschin S. Setty M. Kuo Y.Y. Liu V. Garg V. Sharma R. Simon C.S. Saiz N. Gardner R. Boutet S.C. et al.The emergent landscape of the mouse gut endoderm at single-cell resolution.Nature. 2019; 569: 361-367Crossref PubMed Scopus (113) Google Scholar), an algorithm that allows interrogation of scRNA-seq data across discrete time points (Figures 1D, 1E, and S1D). Force-directed layouts following Harmony implementation ordered cells broadly according to their developmental age (days after conception) (Figure 1D). The day 47 cells were largely separate from other time points, with the exception of an ACTA2HI/TAGLNHI/RGS5+ population of vascular smooth muscle cells (VSMCs) (Muhl et al., 2020Muhl L. Genové G. Leptidis S. Liu J. He L. Mocci G. Sun Y. Gustafsson S. Buyandelger B. Chivukula I.V. et al.Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.Nat. Commun. 2020; 11: 3953Crossref PubMed Scopus (95) Google Scholar; Figures 1D and 1E). Cells were then ordered according to developmental time, with cells from samples older than 101 days (101, 122, 127, and 132 days) clustering together. In addition to the VSMC population, this analysis suggested the emergence of several mesenchymal populations, including a PDGFRAHI/F3HI/DLL1HI population, a GPX3HI population, a TAGLNHI/RGS5− smooth muscle population, and a prominent cluster of cells defined as fibroblasts based on their expression of COLLAGEN genes (COL1A1 and COL1A2) and DECORIN (DCN) (Figures 1E and S1D; Kinchen et al., 2018Kinchen J. Chen H.H. Parikh K. Antanaviciute A. Jagielowicz M. Fawkner-Corbett D. Ashley N. Cubitt L. Mellado-Gomez E. Attar M. et al.Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.Cell. 2018; 175: 372-386.e17Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). F3 has been shown recently to be expressed in a population of mesenchymal cells that is adjacent to the human colonic epithelium (Kinchen et al., 2018Kinchen J. Chen H.H. Parikh K. Antanaviciute A. Jagielowicz M. Fawkner-Corbett D. Ashley N. Cubitt L. Mellado-Gomez E. Attar M. et al.Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.Cell. 2018; 175: 372-386.e17Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), and these cells were additionally characterized by their enrichment of NPY, DLL1, FRZB, and SOX6 (Figures 1E and S1B–S1D). Force-directed layouts following Harmony implementation suggested that different mesenchymal cell populations emerge across developmental time. For example, F3HI/PDGFRAHI cells emerged after approximately 59 days, whereas GPX3HI/F3LO/PDGFRALO and TAGLNHI/RGS5- smooth muscle cells emerge after approximately 80 days. To corroborate the scRNA-seq analysis, we used a combinatorial staining approach, utilizing multiplexed FISH and IF to examine F3 mRNA and SM22, the protein product of the TAGLN gene (Figure 1F). We found that F3 was not expressed at 59 days but was clearly expressed in the villus mesenchyme by 78 days, with expression becoming more restricted to the SECs as time progressed. Interestingly, we observed that F3 was expressed in scRNA-seq at 59 days; however, it is possible that, because fetal tissue staging is an approximation, samples may be slightly older or younger than their actual labeling, explaining slight discrepancies such as this. SM22 was expressed in the day 59 intestine, but only in the outermost muscularis externa layer. SM22 expression in the muscularis mucosa, the layer closest to the intestinal epithelium and adjacent to the proliferative crypt domains, was first observed as poorly organized cells near the epithelium at 100 days that became more organized after this time point. Single-cell analysis and FISH/IF collectively suggest that the mesenchyme in the early fetal intestine is naive and that mesenchymal cell emergence coincides with formation of proliferative intervillus/crypt domains. To understand how mesenchymal cell populations are organized spatially within the tissue after 100 days, we used FISH/IF and confirmed that F3HI cells co-express PDGFRA, DLL1, and NPY (Figure 1G). Interestingly, NPY marked a subset of F3HI SECs lining the villus but was absent from villus SECs (Figures 1G and S2D). GPX3HI/F3LO/PDGFRALO cells were most abundant within the core of intestinal villi and were observed sitting adjacent to NPYHI cells (Figure 1E). It has been demonstrated that several niche factors allow adult and developing human and murine intestinal epithelium to be cultured ex vivo as organoids (Fordham et al., 2013Fordham R.P. Yui S. Hannan N.R.F. Soendergaard C. Madgwick A. Schweiger P.J. Nielsen O.H. Vallier L. Pedersen R.A. Nakamura T. et al.Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury.Cell Stem Cell. 2013; 13: 734-744Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar; Fujii et al., 2018Fujii M. Matano M. Toshimitsu K. Takano A. Mikami Y. Nishikori S. Sugimoto S. Sato T. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition.Cell Stem Cell. 2018; 23: 787-793.e6Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar; Kraiczy et al., 2017Kraiczy J. Nayak K.M. Howell K.J. Ross A. Forbester J. Salvestrini C. Mustata R. Perkins S. Andersson-Rolf A. Leenen E. et al.DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.Gut. 2017; 68: 49-61Crossref PubMed Scopus (72) Google Scholar; Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (3852) Google Scholar, Sato et al., 2011bSato T. Stange D.E. Ferrante M. Vries R.G.J. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1939) Google Scholar; Tsai et al., 2018Tsai Y.-H. Czerwinski M. Wu A. Dame M.K. Attili D. Hill E. Colacino J.A. Nowacki L.M. Shroyer N.F. Higgins P.D.R. et al.A Method for Cryogenic Preservation of Human Biopsy Specimens and Subsequent Organoid Culture.Cell. Mol. Gastroenterol. Hepatol. 2018; 6: 218-222.e7Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These factors often include WNT and RSPO ligands, BMP/transforming growth factor β (TGF-β) antagonists, and EGF and are based on defined growth conditions that allow expansion of intestinal epithelium in vitro (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (3852) Google Scholar, Sato et al., 2011bSato T. Stange D.E. Ferrante M. Vries R.G.J. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1939) Google Scholar). Efforts have been made to determine more physiological niche factors for in vitro culture systems based on observed in vivo niche cues (Fujii et al., 2018Fujii M. Matano M. Toshimitsu K. Takano A. Mikami Y. Nishikori S. Sugimoto S. Sato T. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition.Cell Stem Cell. 2018; 23: 787-793.e6Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar); however, niche factors have not been interrogated in the developing human gut using high-resolution technologies such as scRNA-seq. To identify putative niche factors, we first determined which cells within the human fetal intestine expressed known niche factors. We observed that F3HI/PDGFRAHI SECs and GPX3HI/F3LO/PDGFRALO villus core cells lacked robust expression of most known niche factors (Figures 2A and 2B ), whereas the WNT pathway members with the highest expression were RSPO2, RSPO3, and WNT2B, which are expressed in TAGLNHI/RGS5− smooth muscle cells and COL1A1HI fibroblasts but not expressed by F3HI/PDGFRAHI SECs (Figures 2A and 2B). EGF is a critical driver of proliferation in murine enteroid culture (Basak et al., 2017Basak O. Beumer J. Wiebrands K. Seno H. van Oudenaarden A. Clevers H. Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells.Cell Stem Cell. 2017; 20: 177-190.e4Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar); however, EGF expression was not observed in the mesenchyme, whereas the EGF family member NRG1 was abundant in the F3HI/PDGFRAHI cell population (Figures 2A and 2B). Of note, NRG1 was one of the most robustly expressed EGF family members in the F3HI/PDGFRAHI cell population (Figure S3A). IF for SM22 combined with FISH for RSPO2, RSPO3, WNT2B, EGF, and NRG1, revealed expression patterns that were consistent with scRNA-seq data (Figures 2C and S2F). Given the importance of EGF for in vitro enteroid culture, we further interrogated whether EGF and EGF receptors are expressed in the developing intestinal epithelium via scRNA-seq and FISH. All epithelial cells were extracted and re-clustered, and the data were visualized using a force-directed layout following application of Harmony (Figure 2D). ERBB receptors, including EGFR, ERBB2, and ERBB3, were broadly expressed throughout the epithelium, a finding that was confirmed by FISH, whereas ERBB4 was not expressed (Figures 2E and 2F). Although EGF is not expressed in the intestinal mesenchyme (Figures 2A–2C), we observed that EGF is expressed in a small subset of differentiated epithelial FABP2HI enterocytes (Figure 2G), a finding that was supported using co-FISH/IF and showed that EGF expression is low/absent from the proliferative crypt domain but expressed several cell diameters above the MKI67+ crypt region and throughout the villus epithelium (Figure 2D). On the other hand NRG1 is expressed in F3HI/PDGFRAHI SECs adjacent to the crypt (Figures 2C, 2H, and S2B). Based on the expression pattern of NRG1, we hypothesized that it may act as an ERBB niche signaling cue and may be physiologically relevant in vitro based on its localization and proximity to ISCs within the developing intestine in vivo. To interrogate the effects of NRG1 and EGF on the intestinal epithelium, we split established human fetal duodenum-derived epithelium-only intestinal enteroids (established from a 142-day specimen) in culture using standard growth conditions (WNT3A/RSPO3/NOG plus EGF; STAR Methods) into two groups. One group of enteroids was cultured in standard medium with EGF (100 ng/mL), and the other was grown without EGF and was instead supplemented with NRG1 (100 ng/mL) (Figure 3A). Following growth for 5 days in EGF or NRG1, enteroids did not appear phenotypically different (Figure 3B). Upon interrogation using IF and FISH, we observed that EGF-grown cultures had OLFM4+ and OLFM4− enteroids and that enteroids in this group were highly proliferative based on MKI67 staining (Figure 3C). NRG1-treated enteroids appeared to have more uniform OLFM4 expression but also had fewer KI67+ cells per field of view. To more closely interrogate these differences, each group was subjected to scRNA-seq to investigate transcriptional differences. To reduce any chances of batch effect, all processing for single-cell sequencing for these groups was carried out at the same time in parallel, libraries were prepared in parallel, and samples were sequenced on the same lane (see STAR Methods for details). Despite varying only EGF or NRG1 in the culture, we observed a difference in gene expression between the two groups, as visualized in UMAP plots illustrated by nearly completely independent clustering of cells by culture medium composition (Figures 3D and 3E). The exception to this was cluster 4, which expressed proliferation markers (MKI67 and TOP2A) and had a contribution from both samples (Figures 3E and 3F). Cluster 4 appeared to have a higher number of cells from the EGF-gro