Human pancreas organoid_DMSO
Homo sapiens
Protocols: 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.  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 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.
ENA first public
2026-01-21
INSDC center name
MPI-CBG
INSDC status
public
Submitter Id
E-MTAB-16105:Human pancreas organoid_DMSO
broker name
ArrayExpress
collection date
not collected
common name
human
geographic location (country and/or sea)
not collected
growth condition
PE medium
isolate
not applicable
organism part
pancreas
sample name
E-MTAB-16105:Human pancreas organoid_DMSO
scientific_name
Homo sapiens