Stem cell-derived mesoderm and pancreatic progenitor 3D culture mimics mouse embryonic pancreas and its endocrine compartment
Description
The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aimed to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPS) and pancreas progenitors (PPs), without using extracellular matrix substitutes. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP+MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP+MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development.
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On day 22 (day 8 AA), PP+MP aggregates (2 biological replicates), PP aggregates and PP+Matrigel were dissociated into single cells. PP+MP and PP aggregates were dissociated with accutase, whereas PP+Matrigel were dissociated with TrypLE Select 1X (Gibco). A 10x Genomics single-cell transcriptomic service was used to sequence the cells of the aggregates. For batch 1, a single-cell RNA sequencing library was generated for 3,121 cells from PP+MP aggregates, while for batch 2, three libraries were generated for 6,602 cells of PP+MP aggregates, 7,575 cells of the PP aggregate and 4,240 cells of the PP+Matrigel sample. The libraries were prepared at the Technion Genomics Centre, according to the 10x manufacturer’s protocol (Chromium Next GEM Single Cell 3’ Library & Gel Bead Kit v3.1, PN-1000121), using 7,000 and 15,000 input cells per sample for batch 1 and batch 2, respectively. Single-cell separation was performed using the Chromium Next GEM Chip G Single Cell Kit (PN-1000120). The RNA-seq data was generated on Illumina NextSeq2000, P2 100 cycles (Read1-28; Read2-90; Index1-10; Index2-10) (Illumina, cat no. 20046811). Cell Ranger version 6 and version 7.1 (for batch 1 and batch 2 respectively, 10x Genomics) was used to process raw sequencing data and the Seurat R package version 4.3.0 was used to build the expression matrix. Gene expression was quantified by unique molecular identifiers (UMI) counts.