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Examination of the role of BMP signaling in patterning of human pluripotent stem cell derived mid/hindgut spheroids and maintenance of this patterning in organoids transplanted under mouse kidney capsules.

ABSTRACT: Examination of the role of BMP signaling in patterning of human pluripotent stem cell derived mid/hindgut spheroids and maintenance of this patterning in organoids transplanted under mouse kidney capsules.

PROVIDER: PRJEB20378 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR1923800.fastq.gz	Fastqsanger.gz
	ERR1923810.fastq.gz	Fastqsanger.gz
	ERR1923820.fastq.gz	Fastqsanger.gz
	ERR1923801.fastq.gz	Fastqsanger.gz
	ERR1923811.fastq.gz	Fastqsanger.gz

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Examination of the role of BMP signaling in patterning of human pluripotent stem cell derived mid/hindgut spheroids and maintenance of this patterning in organoids transplanted under mouse kidney capsules.

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.

2017-04-20 | E-MTAB-5658 | biostudies-arrayexpress

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Project description:For definitive endoderm differentiation, iPS cells (day0) were treated with 100 ng ml-1 activin A and 3μM CHIR99021 for 1 day and 100 ng ml-1 activin A for the following two days. Definitive endoderm was subsequently treated with 3uM CHIR99021 and 500ng ml-1 FGF4 for mid/hindgut differentiation. Mid/hindgut floating spheroids (fl; t-Spheroids) were collected from culture medium from day6 to day 8. On day6, mid/hindgut cells were dissociated into single cells and seeded onto EZSPHERE plate to generate suspension spheroids(EZ; s-Spheroids). RNA of fl spheroids and EZ spheroids were extracted using QIAGEN RNeasy kit.

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Project description:Purpose: Combination of systemic RNAi with RNAseq to identify target genes of important pathways in early development Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function.

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