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RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [single cell RNA-Seq]

ABSTRACT: RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [single cell RNA-Seq]

PROVIDER: PRJNA510945 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8358601_1.fastq.gz	Fastqsanger.gz
	SRR8358601_2.fastq.gz	Fastqsanger.gz
	SRR8358602_1.fastq.gz	Fastqsanger.gz
	SRR8358602_2.fastq.gz	Fastqsanger.gz
	SRR8358603_1.fastq.gz	Fastqsanger.gz

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RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [bulk RNA-seq]

Project description:RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [bulk RNA-seq]

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RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [bulk RNA-seq]

Project description:The Recombination Activation Gene, RAG1, expression of which presages T-cell receptor gene rearrangement, is a key marker of T-cell commitment. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cells genesis in the context of haemtopoietic organoids. We show that T-cell commitment occurs concomitantly with the emergence of blood cells from AGM-like haemogenic endothelium, predating the surface expression of CD5 and CD7. In this system, RAG1 marks an early haematopoietic progenitor emerging from SOX17+ endothelium, prior to down regulation of CD90 and VCAM and upregulation of the blood cell marker, CD45. Sort and re-culture experiments show that early RAG1+ cells possess T-cell, B-cell, myeloid and erythroid potential. However, under conditions that favor T-cell development, early RAG1+ cells progress to the CD4+CD8+CD3+ stage, vindicating their classification as bone fide T-cell progenitors. These observations suggest that like the zebrafish and mouse, humans can execute a non-HSC derived wave of T-cell development that includes a multipotent RAG1+ progenitor.

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RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [single cell RNA-Seq]

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Project description:To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar proteins in a manner dependent on amino acids 216-383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increases in V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, which are required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.

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