Congruence of location-specific transcriptional programs in intestinal organoids during long-term culture

Abstract

The emergence of intestinal organoids, as a stem cell-based self-renewable model system, has led to many studies on intestinal development and cell-cell signaling. However, potential issues regarding the phenotypic stability and reproducibility of the methodology during culture still needs to be addressed for different organoids. Here we investigated the transcriptomes of intestinal organoids derived from the same pig as well as batch-to-batch variation of organoids derived from different pigs over long-term passage. The set of genes expressed in organoids closely resembled that of the tissue of origin, including location specific functions, for at least 17 passages. Minor differences in gene expression were observed between individual organoid cultures. In contrast, most tissue-specific genes were not expressed in the transformed jejunum cell line IPECJ2, which also showed gene expression consistent with cancer phenotypes. We conclude that intestinal organoids provide a robust and stable model for translational research with clear advantages over transformed cells. Methods Intestinal organoid generationJejunum tissue segments were obtained from control piglets used for another study, following guidelines of the animal ethics committee of Wageningen University. Two 5-week-old piglets were used for generating organoids following procedures previously described [11,13]. Briefly, a 2 cm section of the mid-jejunum was dissected and placed in ice-cold PBS. After opening the sections longitudinally, jejunum segments were washed three times in ice-cold PBS and villi removed by carefully scraping with a scalpel. Small sections of the mucosa were cut from the muscle layer, divided into small cubes, and transferred into ice-cold PBS containing 30 mM EDTA and incubated with rotation at room temperature for 15 min. The PBS -EDTA was then replaced and incubation continued for 10 min at 37 °C. After washing in ice-cold DMEM supplemented with 5% penicillin/streptomycin (Gibco, Thermo Fisher Scientific), the crypts were dissociated by rigorous vortexing and passed through a 100 µm strainer into cold DMEM containing 5% foetal bovine serum (FBS, v/v). Crypts were pelleted by centrifugation at 300 x g for 5 min, and suspended in Matrigel (Basement Membrane, Growth factor reduced, REF 356231, Corning, Bedford, MA, USA). To improve surface tension, empty 24-well plates were pre-incubated overnight at 37 °C. Matrigel containing crypts was then plated at 7 domes per well (approx. 35 µl per well) and inverted to polymerize at 37 °C. After polymerization, 600 µl F12 cell culture medium (Gibco) was added, supplemented with 100 μg/ml primocin (Invivogen), 10 mM HEPES (HyClone), 1 × B-27 (Gibco), 1.25 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D systems), 15 nM gastrin, 10 mM nicotinamide, 10 μM p38 MAPK inhibitor (Sigma), 600 nM TGFβ receptor inhibitor A83-01, and conditioned media for recombinant Noggin (15% v/v), Spondin (15% v/v), and Wnt3A (30% v/v) provided by dr. Kuo and Hubrecht Institute (Utrecht, the Ne...

Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers

“…1). To verify that our 2D monolayers of organoid cells contained heterotypic epithelial cell lineages as previously reported [8], we performed histological and transcriptomic analyses. Our 2D monolayers indeed contained secretory Paneth cells, and mucus-positive goblet cells as shown by immunohistochemical staining (Fig.…”

“…The mice were housed together in a specific pathogen-free environment with ad libitum access to a standard diet (AIN-93 M) and water. Mice were euthanized and dissected to remove the duodenum and the crypts isolated for generating organoids as previously described with minor modifications [7,8] and grown in basal culture medium (W-ENR: DMEM/F12 medium enriched with 50 ng/mL mouse Epidermal growth factor (mEGF), 10 mmol/L Hepes (Invitrogen, the Netherlands), 1× B-27 supplement (ThermoFisher scientific, The Netherlands), and recombinant Noggin (15% v/v), WNT3A (30% v/v, Hubrecht Institute, Utrecht, Netherlands [7]), and R-Spondin (15% v/v, Stanford university, Palo Alto, USA [5]) (Method S1). To facilitate access to the apical surface of epithelial cells, we generated 2D monolayers of cells from dissociated 3D organoids.…”

“…To overcome these issues, we used three-dimensional (3D) organoid cultures from isolated crypts of the duodenum [5,6]. Intestinal organoids can be generated within 2 weeks, maintained for > 2 years in culture or cryopreserved, and when differentiated contain all the epithelial cell lineages found at the location of origin [7,8]. Recently, we and others have described methods for growing polarised monolayers of 3D organoid cells on semipermeable membrane supports, enabling apical exposure to luminal factors [9][10][11].…”

Congruence of Transcription Programs in Adult Stem Cell-Derived Jejunum Organoids and Original Tissue During Long-Term Culture

“…This manuscript has been released as a pre-print at bioRxiv by Van Der Hee et al (2019) . We would like to thank Anja Taverne-Thiele, Nico Taverne, and Linda Loonen for their practical assistance and helpful discussions.…”

Immune regulation by human colonic bacteria and short-chain fatty acids

“…Library preparations were performed using total RNA extracted from acetate, butyrate, or medium-treated organoids and then sequenced on an Illumina sequencing system by Novogene (Hong Kong). The sequencing reads were checked for quality using CLC Genomic Workbench (QIAGEN) and FastQC [150] and mapped to the Sus scrofa 11.1 reference genome (Ensembl, [151], see Supplementary Table S6.1), and processed as previously described [152] for further downstream analysis. Ileum organoids were incubated with acetate, butyrate or medium control for 5 hours, followed by RNA extraction and library preparation.…”