Gene epression profile of s-spheroids and t-spheroids differentiated from iPS cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE182230 | GEO | 2022/11/15
REPOSITORIES: GEO
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Project description:Human lung organoids (HLOs) were derived from human embryonic stem cell line H9 NIH registry #0062. Sequencing of HLOs was performed by the UM DNA Sequencing Core, using Illumina Hi-Seq platform and single-end 50 bp reads. The UM Bioinformatics Core downloaded the reads files from the Sequencing Core storage, and concatenated those into a single .fastq file for each sample. 3 HLOs were cultured for 65 days and 3 HLOs were cultured in vitro for 110 days. HLO Culture Protocol 4-day Activin A (R&D systems) differentiation protocol was used to derive definitive endoderm from human pluripotent stem cells (hPSCs). Cells were treated with Activin A (100ngml-1) for three consecutive days in RPMI 1640 media (Life Technologies) with increasing concentrations of 0%, 0.2% and 2% HyClone defined fetal bovine serum (dFBS, Thermo Scientific). The fourth day was a repeat of day 3 media (2% HyClone FBS). After differentiation into definitive endoderm, cells were incubated in foregut media (advanced DMEM/F12 plus N-2 and B27 supplement, 10mM Hepes, 1x L-Glutamine (200mM), 1x Penicillin-streptomycin (5,000 U/mL, all from Life Technologies)) with 200ng/mL Noggin (NOG, R&D Systems) and 10uM SB431542 (SB, Stemgent) for 4 days, SB, 500ng/mL FGF4 (R&D Systems), and 2 uM CHIR99021 (Chiron, Stemgent), and 1uM SAG (Enzo Life Sciences) for 4-6 days. After 4 days with treatment of growth factors, three-dimensional floating spheroids were present in the culture. Three-dimensional spheroids were transferred into Matrigel to support 3D growth. Spheroids were embedded in a droplet of Matrigel (BD Bioscience #356237) in one well of a 24 well plate, and incubated at room temperature for 10 minutes. After the Matrigel solidified, foregut media with 1% Fetal bovine serum (FBS, CAT#: 16000-044, Life Technologies) and 500ng/mL FGF10 (R&D Systems) were overlaid and replaced every 4 days. Organoids were transferred into new Matrigel droplets every 10 to 15 days.
2015-03-09 | E-MTAB-3339 | biostudies-arrayexpress
Project description:Denunded HUVEC/HUASMC spheroids vs. HUASMC spheroids
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Project description:Background: Mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC), named EMSC here, have been found efficacious in animal models of autoimmune, inflammatory, and degenerative diseases. However, all the EMSC derivation methods reported so far are in two-dimensional (2D) culture systems, which are of low efficiency and high cost, difficult for large-scale production for research and therapeutic applications. Methods: We established a 3D system that allowed differentiation of hESC spheroids into MSC spheroids (EMSCSp) following treatment with BMP4 and A8301 for 5 days and subsequent culture in a MSC medium for about 15 days. All the procedures were conducted in one vessel without intermediate passaging. Results: EMSCsp cells were efficiently derived from hESC spheroids within 20 days in the 3D culture system, which could be scaled up from a small culture vessel to a 100-ml plastic bag. EMSCSp could further differentiate into spheroids of chondrocytes or adipocytes. EMSCSp could also reattach and form a 2D-monolayer culture (EMSCSp-ML). Compared to EMSC differentiated in monolayer, EMSCSp-ML had faster proliferation and higher yield, and developed less apoptosis and slower senescence. EMSCSp-ML also retained immune-modulatory effects in vitro and therapeutic effects on two mouse models of colitis. Conclusions: The 3D method provides a simple and economic system for large-scale production of EMSC as an unlimited source of the therapeutically promising cells.
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Project description:The goal of this study was to test the hypothesis that BMP signaling regulates patterning of human CDX2+ gut tube cultures (Spence et al 2011, Watson et al. 2014). The study is comprised of 2 separate experiments. The first experiment was to determine the immediate impact of BMP signaling on CDX2+ gut tube cultures. To do so we tested spheroids, spheroids after plating in Matrigel and exposed to 3 days of Noggin (100ng/mL), spheroids after plating in Matrigel and exposed to 3 days of EGF alone (100ng/mL, Control), and spheroids after plating in Matrigel and exposed to 3 days of BMP2 (100ng/mL). RNA was collected from spheroids and spheroids after 3 days of patterning. The second experiment examined if patterning was maintained after this initial 3 days of patterning and an addition 8-10 weeks following transplantation in vivo. To do this we grew all Matrigel plated spheroids for an additional 25 days in media containing EGF alone (Control media). We then transplanted these organoids under the mouse kidney capsule and allowed them to mature for 8-10 weeks. We then collected RNA from the transplanted organoids.
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