Microarray of 3D cultured basal cells in the presence of Wnt3A. Mus musculus

Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. We show that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We propose here that Wnt3A treated mammary stem cells retain their stemness through the regulation of its downstream target genes. We used microarrays to detail the global gene expression pattern underlying mammary stem cells response towards Wnt3A treatment and identified distinct classes of genes during this process Overall design: Mammary glands from 8- to 12-week-old virgin female mice were isolated and single-cell suspension was obtained. Mammary stem cell enriched population (Lin-, CD24+, CD29hi) cells were isolated using BD FACSAria. The purity of sorted population was routinely checked and ensured to be more than 95%.Total RNA from 2nd colonies passage cultured in the presence of vehicle and Wnt3A was extracted with PicoPure (Arcturs) in accordance with the manufacturer’s protocol. RNA concentration was determined with NanoDrop ND-1000, and quality was determined using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Affymetrix microarray analysis, fragmentation of RNA, labelling, hybridization to Mouse Genome 430 2.0 microarrays, and scanning were performed in accordance with the manufacturer’s protocol (Affymetrix). Total 4 samples representing two sets of replicates were analyzed.