{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Anne Grapin-Botton"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16105"],"description":["To investigate signaling pathways regulating human pancreas differentiation and morphogenesis, we developed a high-content image-based screen and quantitative multivariate analysis pipelines robust to heterogeneity to extract single-cell and organoid features using pancreatic progenitor organoids. Among the 54 hit compounds, we found that GSK3A/B inhibition via WNT signaling has a global reversible effect on cell identity, repressing pancreatic progenitor markers and inducing a poised state of progenitors transitioning to acinar cells. We show that additional FGF repression enables further differentiation of acinar cells recapitulating pancreatic acini morphogenesis and functions . This dataset compares human pancreas organoids produced from pluripotent stem cells by RNA-Seq in expansion conditions in the presence of DMSO (control) to those exposed for 7 days to CHIR99021 or CHIR99021 in the absence of the FGF2 normally included in the expansion medium."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Library sequencing  Libraries were sequenced on an Illumina NovaSeq 6000 (NovaSeq S4 v1.5, 4XP, 200 cycles). Base calling was performed with bcl2fastq2 (v2.20.0). Sequencing yielded 930,222,396 (DMSO), 857,521,610 (CHIR), and 699,688,065 (CHIR noFGF2) reads per sample.","Nucleic Acid Extraction - On day 10, organoids were dissociated into single cells using TrypLE, and cells were sorted into single cells by FACS based on cell viability staining by DAPI separately for each condition, DMSO, CHIR99021 or CHIR99021 in the absence of FGF2, for experiments conducted side-by-side. The viability of single-cell suspensions was 90% (DMSO), 80% (CHIR), and 95% (CHIR noFGF2). For each condition, 27,000 cells were loaded onto a 10x Genomics Chromium GEM-X microfluidic chip (Single Cell 3’ GEM-X, v4 chemistry), targeting ~20,000 captured cells per sample. The Chromium GEM-X Single Cell Gene Expression v4 upgrades short read sequencers to deliver a scalable microfluidic platform for 3' digital gene expression by profiling 500-20,000 individual cells per sample. A pool of ~3,600,000 10x Barcodes are sampled separately to index each cell’s transcriptome. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA (from polyadenylated mRNAs) share a common 10x Barcode. Dual indexed libraries are generated and sequenced from the cDNA, and 10x Barcodes are used to associate individual reads back to the individual partitions.  GEMs are generated by combining barcoded Gel Beads, a Master Mix containing cells, and Partitioning Oil B onto GEM-X 3' Chip. To achieve single cell resolution, cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell, while the remainders largely contain a single cell. Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any copartitioned cell is lysed.","Library Construction - Library contruction  For each condition, 27,000 cells were loaded onto a 10x Genomics Chromium GEM-X microfluidic chip (Single Cell 3’ GEM-X, v4 chemistry), targeting ~20,000 captured cells per sample. cDNA amplification was performed for 10 cycles, followed by 0.6× SPRI purification.","Sample Collection - Generation of pancreas organoids and collection in the three treatment conditions  Expansion and maintenance of human pluripotent stem cells (hPSCs)   The H1 hESC lines was obtained from WiCell. The hPSCs were expanded and maintained in mTeSR1medium (Stem Cell Technologies) on hESC-qualified Matrigel (Corning). Cells were maintained at 37°C and 5% CO2, and the medium was changed daily. Upon reaching around 80% confluency, cells were dissociated with TrypLE (Thermo Fisher Scientific), counted with a cell counter (Countess II FL), and seeded at a density of 40,000 cells/cm2. While passaging, the cells were supplemented with 10 µM ROCK inhibitor Y27632 (Cell Signaling) for the first 24 hours. The cells were regularly tested for Mycoplasma-free status. Differentiation of hPSCs into pancreatic progenitor (PP) cells The step-wise protocol for differentiation based on the publication by Rezania et al 1 was modified and adapted by us2. Briefly, hPSCs were passaged and cultured in 2D on plates coated with growth factor-reduced (GFR) Matrigel (Corning) at 1/30 dilution in DMEM/F12 Glutamax (Thermo Fisher Scientific). Cells were seeded at a density of 350,000 cells/cm2 in mTeSR1 medium supplemented with 10 µM ROCK inhibitor for 24 hours before starting treatment with the differentiation media. Media changes were performed daily. The following media were used for differentiation. Stage 1 basal medium was composed of 1.5 g/L sodium bicarbonate, 1x Glutamax, 10 U/ml penicillin-streptomycin, 10 mM Glucose, and 0.5% fatty acid free bovine serum albumin (FAF-BSA) in MCDB 131 medium. The basal medium was supplemented with 3 µM CHIR and 100 ng/ml Activin A for day 1, 0.3 µM CHIR and 100 ng/ml Activin A for day 2, and 100 ng/ml Activin A for day 3. Stage 2 medium contained 1.5 g/L sodium bicarbonate, 1x Glutamax, 10 U/ml penicillin-streptomycin, 10 mM Glucose, 0.5% FAF-BSA, 0.25 mM ascorbic acid, and 50 ng/ml FGF7 in MCDB 131 medium and was used for 2 days. Stage 3 medium contained 2.5 g/L sodium bicarbonate, 1x Glutamax, 10 U/ml penicillin-streptomycin, 10 mM Glucose, 2% FAF-BSA, 0.25 mM ascorbic acid, 1:200 ITS-X, 1 µM retinoic acid, 0.25 µM Sant-1, 100 nM LDN193189, 200 nM TPB, and 50 ng/ml FGF7 in MCDB 131 medium and was used for 2 days. Stage 4 medium was composed of 2.5 g/L sodium bicarbonate, 1x Glutamax, 10 U/ml penicillin-streptomycin, 10 mM Glucose, 2% FAF-BSA, 0.25 mM ascorbic acid, 1:200 ITS-X, 0.1 µM retinoic acid, 0.25 µM Sant-1, 200 nM LDN193189, 100 nM TPB, and 2 ng/ml FGF7 in MCDB 131 medium and was used for 3 days. At stage 4 day 3, 97% of these cells expressed PDX1, a PP proxy, and 15% expressed NEUROG3, an endocrine progenitor proxy.  Expansion and maintenance of hPSC-derived PP-organoids  Pancreatic progenitor cells at Stage 4 day 3 were harvested using TrypLE and seeded at a density of 40,000 cells in a 40 µl dome of 75% GFR Matrigel (1,000 cells/µl) per well on a 24-well plate. Upon solidification after 10 minute at 37°C, the Matrigel domes were incubated in pancreatic epithelium (PE) medium2 consisting of 1x B27 (Thermo Fischer Scientific), 64 ng/ml FGF2 (Peprotech), and 10 µM ROCK inhibitor in DMEM/F12-Glutamax at 37°C with 5% CO2. Regular medium changes were performed every three days. Embedded in GFR Matrigel, PP cells cluster, proliferate, and self-organize to form PP-organoids. Organoids were passaged every ten days by dissociating into mostly single cells using TrypLE and re-seeding in 75% GFR Matrigel. Using this protocol, PP-organoids can be maintained in long-term cultures including freeze-and-thaw cycles. For quality controls, PP-organoids were regularly tested for expression of PDX1 (>99%) and NKX6-1 (>70%) by flow cytometry and also for Mycoplasma-free status.  Compound treatment  PP-organoids were seeded on day 0 in 75% GFR Matrigel domes and treated with PE medium. On day 3, the organoids were treated with the modified PE medium containing either DMSO, 3 µM CHIR or 3 µM CHIR without FGF2 for seven days. Media changes were performed on days 5, 7, and 9. The spheres were harvested and fixed on day 10 for sequencing.  Collection of samples   On day 10, organoids were dissociated into single cells using TrypLE, and cells were sorted into single cells by FACS based on cell viability staining by DAPI. The viability of single-cell suspensions was 90% (DMSO), 80% (CHIR), and 95% (CHIR noFGF2). For each condition, 27,000 cells were loaded onto a 10x Genomics Chromium GEM-X microfluidic chip.   References  1. Rezania, A., Bruin, J.E., Arora, P., Rubin, A., Batushansky, I., Asadi, A., O'Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T., et al. (2014). Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133. 10.1038/nbt.3033.  2. Goncalves, C.A., Larsen, M., Jung, S., Stratmann, J., Nakamura, A., Leuschner, M., Hersemann, L., Keshara, R., Perlman, S., Lundvall, L., et al. (2021). A 3D system to model human pancreas development and its reference single-cell transcriptome atlas identify signaling pathways required for progenitor expansion. Nat Commun 12, 3144. 10.1038/s41467-021-23295-6."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - normalisation data transformation protocol and further analyses  Reads were demultiplexed and processed with Cell Ranger (v9.0.1, 10x Genomics) using parameters --chemistry=auto and --include-introns=true, aligned to the GRCh38 10x Genomics reference (refdata-gex-GRCh38-2024-A). Cell Ranger invoked Martian Runtime (v4.0.13), Python 3.10.8, and dependencies numpy (v1.26.4), scipy (v1.10.1), pysam (v0.21.0), h5py (v3.9.0), pandas (v2.1.4), and STAR (v2.7.2a). The minimum fraction of valid barcodes was 97.3%, and the fraction of valid UMIs reached 100%. Barcode-rank plots were inspected, and a second Cell Ranger run was executed with manually set expected cell numbers (--force-cells): 21,200 (DMSO), 16,000 (CHIR), and 20,200 (CHIR noFGF2). Mapping quality was high, with ≥93.3% of reads confidently mapped to the genome, ≥85% to the transcriptome, ≤3.6% mapping to intergenic regions, and ≥93.2% reads in cells. The median number of detected genes per cell was ≥4,163. Spliced and unspliced transcript counts were derived using Velocyto (v0.17.17) applied to the Cell Ranger–generated BAM files. Output matrices were stored in MatrixMarket format (v2). Filtered count matrices (filtered_feature_bc_matrix) from the second Cell Ranger run were used for all downstream analyses. Downstream analyses were performed in R (v4.0.5) using Seurat (v4.1.1), SeuratWrappers (v20210208), SingleCellExperiment (v1.12.0), SCTransform (v0.3.5), Scater (v1.18.6), VeloCyto.R (v0.6), scDblFinder (v1.15.1), and DoubletFinder (v2.0.3). Cells were filtered to retain those with ≥750 reads, ≥350 detected genes, and ≤15% mitochondrial read fraction; genes were required to be expressed in ≥3 cells. The mitochondrial gene fraction per cell was calculated from gene annotation. Normalization and variance stabilization were performed with SCTransform, regressing out (1) mitochondrial read fraction, (2) S.Score, and (3) G2M.Score computed by Seurat’s CellCycleScoring function. Normalized data were centered and scaled. No data integration was applied as it erased all biological variation previously observed in bulk sequencing. We note that data integration has been developed with the intent of comparing different populations with overlapping populations of cells where anchors can be easily extracted but not for perturbations leading to large shifts in cell identity. As all three conditions were processed in parallel, low technical noise is expected. This was confirmed by the fact that all endocrine cells from all conditions formed a distinct cluster and that part of the cells in CHIR+No FGF cluster with CHIR alone. Principal component analysis was performed on the 3,000 most variable genes. Cells were clustered using FindClusters (50 iterations, k = 75, entrees = 100, resolution = 0.1), and marker genes were identified with FindAllMarkers (only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25, test. use = \\\"wilcox\\\", ba e = 2) after running PrepSCTFindMarkers. Visualization was performed in Seurat (v5.3.3) and ggplot2 (v3.5.2)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Cell culture","Chromium Controller, 10x Genomics and  Chromium X, 10x Genomics","Chromium Controller,  10x Genomics and Chromium X, 10x Genomics","Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Anne Grapin-Botton"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of human pancreas organoids in expansion conditions in the presence of DMSO, CHIR99021 or CHIR99021 in the absence of FGF2","description":"To investigate signaling pathways regulating human pancreas differentiation and morphogenesis, we developed a high-content image-based screen and quantitative multivariate analysis pipelines robust to heterogeneity to extract single-cell and organoid features using pancreatic progenitor organoids. Among the 54 hit compounds, we found that GSK3A/B inhibition via WNT signaling has a global reversible effect on cell identity, repressing pancreatic progenitor markers and inducing a poised state of progenitors transitioning to acinar cells. We show that additional FGF repression enables further differentiation of acinar cells recapitulating pancreatic acini morphogenesis and functions . This dataset compares human pancreas organoids produced from pluripotent stem cells by RNA-Seq in expansion conditions in the presence of DMSO (control) to those exposed for 7 days to CHIR99021 or CHIR99021 in the absence of the FGF2 normally included in the expansion medium.","dates":{"release":"2026-01-21T00:00:00Z","modification":"2026-05-27T13:34:52.223Z","creation":"2025-11-12T16:55:04.158Z"},"accession":"E-MTAB-16105","cross_references":{"ENA":["ERP184325"],"Biostudies":["E-MTAB-14625"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}