Targeted ablation of Lgr5-expressing intestinal stem cells in diphtheria toxin receptor-based mouse and organoid models

STAR protocols

2022 Jun 17

Lim, HYG;Yada, S;Barker, N;
PMID: 35620071 | DOI: 10.1016/j.xpro.2022.101411

Intestinal cells marked by Lgr5 function as tissue-resident stem cells that sustain the homeostatic replenishment of the epithelium. By incorporating a diphtheria toxin receptor (DTR) cassette linked to the Lgr5 coding region, native Lgr5-expressing cells are susceptible to ablation upon DT administration in vivo. A similar strategy can be used for Lgr5-expressing cells within organoids established from DTR models. Together, these in vivo and in vitro approaches will facilitate dissection of the roles of Lgr5-expressing cells residing in different tissue compartments. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).

Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis.

Nature

2017 Aug 16

Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, Nusse R, Amieva MR, Meyer TF.
PMID: 28813421 | DOI: 10.1038/nature23642

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

Nat Commun

2020 Jan 17

Kim JE Fei L, Yin WC, Coquenlorge S, Rao-Bhatia A, Zhang X, Shi SSW, Lee JH, Hahn NA, Rizvi W, Kim KH, Sung HK, Hui CC, Guo G, Kim TH
PMID: 31953387 | DOI: 10.1038/s41467-019-14058-5

Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development

Developmental cell

2022 Jun 07

Hein, RFC;Wu, JH;Holloway, EM;Frum, T;Conchola, AS;Tsai, YH;Wu, A;Fine, AS;Miller, AJ;Szenker-Ravi, E;Yan, KS;Kuo, CJ;Glass, I;Reversade, B;Spence, JR;
PMID: 35679862 | DOI: 10.1016/j.devcel.2022.05.010

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

An unsupervised method for physical cell interaction profiling of complex tissues

Nature methods

2021 Jul 12

Andrews, N;Serviss, JT;Geyer, N;Andersson, AB;Dzwonkowska, E;Šutevski, I;Heijboer, R;Baryawno, N;Gerling, M;Enge, M;
PMID: 34253926 | DOI: 10.1038/s41592-021-01196-2

Cellular identity in complex multicellular organisms is determined in part by the physical organization of cells. However, large-scale investigation of the cellular interactome remains technically challenging. Here we develop cell interaction by multiplet sequencing (CIM-seq), an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between cell types present in a tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution into constituent cell types. CIM-seq estimates parameters such as number of cells and cell types in each multiplet directly from sequencing data, making it compatible with high-throughput droplet-based methods. When applied to gut epithelium or whole dissociated lung and spleen, CIM-seq correctly identifies known interactions, including those between different cell lineages and immune cells. In the colon, CIM-seq identifies a previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is directly adjacent to colonic stem cells. Our results demonstrate that CIM-seq is broadly applicable to unsupervised profiling of cell-type interactions in different tissue types.

Charting human development using a multi-endodermal organ atlas and organoid models

Cell

2021 May 17

Yu, Q;Kilik, U;Holloway, EM;Tsai, YH;Harmel, C;Wu, A;Wu, JH;Czerwinski, M;Childs, CJ;He, Z;Capeling, MM;Huang, S;Glass, IA;Higgins, PDR;Treutlein, B;Spence, JR;Camp, JG;
PMID: 34019796 | DOI: 10.1016/j.cell.2021.04.028

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions

PLoS One

2020 Apr 17

Ambrosini YM, Park Y, Jergens AE, Shin W, Min S, Atherly T, Borcherding DC, Jang J, Allenspach K, Mochel JP, Kim HJ
PMID: 32302323 | DOI: 10.1371/journal.pone.0231423

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BMC Biol.

2019 Apr 11

Chandra L, Borcherding DC, Kingsbury D, Atherly T, Ambrosini YM, Bourgois-Mochel A, Yuan W, Kimber M, Qi Y, Wang Q, Wannemuehler M, Ellinwood NM, Snella E, Martin M, Skala M, Meyerholz D, Estes M, Fernandez-Zapico ME, Jergens AE, Mochel JP, Allenspach K.
PMID: 30975131 | DOI: 10.1186/s12915-019-0652-6

Abstract

BACKGROUND:

Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures.

RESULTS:

Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research.

CONCLUSIONS:

We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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