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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm

ABSTRACT: Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm

PROVIDER: PRJNA432522 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6512540_1.fastq.gz	Fastqsanger.gz
	SRR6512540_2.fastq.gz	Fastqsanger.gz
	SRR6512550_1.fastq.gz	Fastqsanger.gz
	SRR6512550_2.fastq.gz	Fastqsanger.gz
	SRR6512541_1.fastq.gz	Fastqsanger.gz

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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm [RNA-Seq]

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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm [RNA-Seq]

Project description:Differentiation of human pluripotent stem cells toward definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.

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Project description:HESC-H9 and iPSC lines 3.5, 3.6 and 3.12 were analyzed using Affymetrix microarray before and after Definitive Endoderm (DE) formation. DE was induced using the ActivinA differentiation protocol described by D'Amour et al., 2006 (PMID: 16258519) Clustering analysis of transcripts that were differentially regulated during DE formation indicated that iPSC lines 3.5 and 3.12 differentiate in manner that is highly similar to HESC-H9 cells iPSC line 3.6 had a more divergent transcriptional profile. Three induced pluripotent stem cell lines (iPSC) and one human embryonic stem cell line (hESC - H9) were collected as undifferentiated (UD) cells, and flash frozen. These cell lines were also subjected to definitive endoderm (DE) induction, collected and flash frozen. RNA was harvested from the frozen cell pellets and hybridized to the Affymetrix microarray chip. The three iPSC cell lines are iPSC 3.5, iPSC 3.6. and iPSC 3.12. In the UD state, iPSC 3.5 was analyzed in duplicate, while iPSCs 3.6 and 3.12 and H9s were analyzed in biological triplicate. All four cell lines were analyzed as biological triplicates for DE induction.

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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm

Project description:This SuperSeries is composed of the SubSeries listed below.

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