biostudies-arrayexpressCharlie ChildsHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13469Here, we used single-nuclei RNA-sequencing (snRNA-seq) to profile pluripotent stem cell-derived human intestinal organoids (HIOs) grown in media comprised of minigut media + 10 ng/ml EREG in vitro for 28 days and transplanted into the kidney capsule of a mouse for 12 weeks.biostudies-arrayexpressSequencing - All single-cell RNA-sequencing was performed with an Illumina NovaSeq 6000 by the University of Michigan DNA Sequencing core.Growth Protocol - Maintenance and differentiation into HIOs were carried out as previously described. Cells were kept in a 37°C tissue culture incubator with 5% CO2 and lines were maintained in mTeSR Plus cultured media (Stemcell Technologies Cat# 100-1130). Stem cells underwent directed differentiation into definitive endoderm over a 3-day treatment using Activin A (100ng/mL, R&D Systems Cat#338-AC) added to RPMI base media. This base media was supplemented with 0%, 0.2%, 2% HyClone dFBS (Thermo Fischer Cat#SH3007103) on subsequent days with the addition of 5 mL penicillin-streptomycin each day (Gibco Cat# 15070063). After three days, endoderm monolayers were differentiated into an intestinal identity by treatment with FGF434 (500ng/mL) and CHIR99021 (2μM, APExBIO Cat#A8396). On days 4-6 of hindgut differentiation, spheroids budded from the monolayer and were collected. These spheroids were embedded in Matrigel as previously described32 and maintained in basal growth media consisting of Advanced DMEM/F12 (Gibco Cat# 11320033) with B27 (50x, Thermo Fisher Cat#17504044), GlutaMAX (1X, Gibco Cat#35050061), penicillin-streptomycin (Gibco Cat# 15070063), and HEPES buffer (15 mM , Gibco Cat#15630080). Organoid basal growth media was supplemented with epidermal growth factor (EGF) (100 ng/mL, 10 ng/mL, 1 ng/mL R&D Systems Cat#236-EG-01M) or Epiregulin (EREG) (100 ng/mL, 10 ng/mL, 1 ng/mL R&D Systems Cat#1195-EP-025/CF) with Noggin-Fc (100ng/mL, purified from conditioned media35), and R-Spondin1 (5% conditioned medium36) for the first three days of culture to pattern a proximal small intestine. On the third day after embedding, media was changed to basal growth media supplemented with EGF or EREG only (no additional Noggin or R-Spondin1) and remained in this media for the duration of the experiments with media changes every 5 days. Organoids were not passaged to avoid disrupting the development and spatial organization of the key cell types seen in EREG-grown HIOs. After at least 28 days, organoids were transferred into the kidney capsule of a mouse and allowed to grow for 10-12 weeks before harvesting.Nucleic Acid Extraction - Nuclei were isolated and permeabilized in accordance with 10x Genomics’ Chromium Nuclei Isolation Kit Protocol (10x Genomics Cat#1000493). Briefly, tissue was minced into smaller fragments and then placed in lysis buffer where it was further dissociated mechanically with a pellet pestle. Tissue was then incubated in the lysis buffer for 5-7 minutes. The suspension was passed through the nuclei isolation column and spun at 16,000g for 20 seconds at 4°C. The suspension was then vortexed for 10 seconds and centrifuged at 500g for 3 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 500 µL of Debris Removal Solution and centrifuged at 700g for 10 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 1 mL of Wash Solution and centrifuged at 500g for 5 minutes at 4°C twice. The final pellet was resuspended in diluted nuclei buffer. Nuclei capture was carried out on the 10X Chromium platform with a target capture of 5000 nuclei per sample, and libraries were immediately prepared by the University of Michigan Advanced Genomics Core facility.Library Construction - Nuclei capture was carried out on the 10X Chromium platform with a target capture of 5000 nuclei per sample, and libraries were immediately prepared by the University of Michigan Advanced Genomics Core facility.Sample Collection - Organoids were retrieved from their cultures or from their murine host and immediately snap-frozen for future nuclear extraction.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesUnknownTranscriptomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensCoordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.Charlie ChildsJason SpencefalsesnRNA-seq of pluripotent stem cell-derived human intestinal organoids grown in vitro and in vivo with EpiregulinHere, we used single-nuclei RNA-sequencing (snRNA-seq) to profile pluripotent stem cell-derived human intestinal organoids (HIOs) grown in media comprised of minigut media + 10 ng/ml EREG in vitro for 28 days and transplanted into the kidney capsule of a mouse for 12 weeks.2026-01-19T00:00:00Z2026-01-19T14:37:14.068Z2026-01-19T14:36:45.424ZE-MTAB-13469ERP187869EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0005518EFO_0004184