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Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells

ABSTRACT: Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells

PROVIDER: PRJNA603452 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10973200.fastq.gz	Fastqsanger.gz
	SRR10973300.fastq.gz	Fastqsanger.gz
	SRR10973400.fastq.gz	Fastqsanger.gz
	SRR10973500.fastq.gz	Fastqsanger.gz
	SRR10973201.fastq.gz	Fastqsanger.gz

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Project description:Molecular distinctions between the stasis and telomere attrition senescence barriers in cultured human mammary epithelial cells Normal human epithelial cells in culture have generally shown a limited proliferative potential of ~10-40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in media composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50-60 population doublings, followed by p16(+), senescence-associated b-galactosidase(+) stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, i.e., cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo, in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean TRF length, and genomic stability, differed significantly between HMEC populations at the stasis vs. telomere attrition senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere attrition, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is Rb-mediated and independent of telomere length, while a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multi-lineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC. 48 samples from Human Mammary Epithelial cells which includes samples from four different individuals at different passage levels which includes prestasis,intermediate,post selection and agonesence stages of cell cycle.

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Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells

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