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Single-cell RNA sequencing of mammary epithelial and myoepithelial cells

ABSTRACT: Single-cell RNA sequencing of mammary epithelial and myoepithelial cells

PROVIDER: PRJNA1173731 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31016515_1.fastq.gz	Fastqsanger.gz
	SRR31016515_2.fastq.gz	Fastqsanger.gz
	SRR31016516_1.fastq.gz	Fastqsanger.gz
	SRR31016516_2.fastq.gz	Fastqsanger.gz
	SRR31016517_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA sequencing of mammary epithelial and myoepithelial cells

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Project description:There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in DFCI-1 medium retain a fraction with progenitor cell properties. These cells co-express basal, luminal and stem/progenitor cell markers. Clonal derivatives of progenitors co-expressing these markers fall into two distinct types: K5+/K19- (Type I) and K5+/K19+ (Type II). We show that both types of progenitor cells have self-renewal and differentiation ability. Through microarray analysis, we want to identify genes and pathways linked to human mammary epithelial stem/progenitor cell self-renewal and differentiation. Normal human mammary epithelial cells (hMECs) were isolated from Reduction Mammoplasty and immortalized by hTERT. Type I K5+/K19- and Type II K5+/K19+ cell colonies were isolated from hTERT-immortalized hMECs and cultured in MEGM medium for self-renewal and differentiation. Total RNA isolated from Type I, Type II, and differentiated Myoepithelial (Myo) cells were used on Affymetrix microarrays.

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Project description:To get molecular insight into age- and compartment-specific changes in telomere maintenance and associated properties in human mammary gland, we analyzed distinct subsets of normal human mammary epithelial cells. The cells were isolated by fluorescent activated cell sorting (FACS) directly from mammary tissue obtained from normal women undergoing reduction mammoplasties with concomitant removal of hematopoietic and endothelial cells by depletion of CD45pos and CD31pos cells. The three epithelial cell populations then isolated were: (i) CD49fhiEPCAMneg/low cells, (ii) CD49fposEPCAMpos cells and (iii) CD49fnegEPCAMpos cells. The CD49fhiEPCAM-/low cells are selectively enriched in mammary stem cells with functional mammary gland regenerating activity in suitably transplanted immunodeficient mice, bipotent progenitors that form colonies of adherent myoepithelial and luminal cells in vitro, myoepithelial-restricted progenitors that form colonies of exclusively adherent myoepithelial cells in vitro, and mature myoepithelial cells that are not clonogenic (collectively referred to as basal cells, BCs). The CD49fposEPCAMpos cells are selectively enriched in luminal progenitors (referred to as luminal progenitors, LPs); and the CD49fnegEPCAMpos cells are selectively enriched in mature luminal cells (referred to as luminal cells, LCs). Differences in gene expression in general and telomere associated genes in particular were elucidated by analyzing mammary epithelial subpopulations. Total RNA was isolated from 24 samples obtained from FACS purification of mammary epithelial subpopulations from 9 reduction mammoplasty breast tissues. Global gene expression profiling was performed by array.

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