Project description:To understand how Wnt signaling affects AM phenotype in vitro, we treated AMs pooled from 5 WT mice with or without Wnt3a for overnight and performed single-cell RNA sequencing analysis in the cells. Dimensionality reduction analysis of combined control or Wnt3a-treated AMs revealed eight clusters based on their gene expression pattern, and control or Wnt3a-treated AMs were almost mutually exclusive as visualized in UMAP plot.
Project description:To identify molecular characteristics of WT AM without Wnt3a, and WT, b-catenin-deficient or HIF-1a-deficient AM with Wnt3a stimulation in vitro, we isolated RNA from AMs with various treatments and examined by bulk RNA-seq. We found a large number of gene profiles were altered following Wnt3a treatment in WT AMs, while b-catenin and HIF-1a deficiency largely abolished the effects of Wnt3a treatment on AM transcription, suggesting that b-catenin and HIF-1a mediates most downstream effects of Wnt3a in AMs. Indeed,HIF-1a-deficient AMs essentially phenocopied those of b-catenin-deficient AMs, with more than 80% of differentially expressed genes in HIF-1a-deficient AMs overlapping with b-catenin-deficient AMs following Wnt3a treatment.
Project description: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 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 manufacturerM-bM-^@M-^Ys 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 manufacturerM-bM-^@M-^Ys protocol (Affymetrix). Total 4 samples representing two sets of replicates were analyzed.
Project description:We investigated whether in vitro expansion of human alveolar epithelial type II cells is possible. We found that human endogenous human alveolar epithelial type II cells can be cultured and passaged. The culture system enabled retroviral gene transduction into human alveolar epithelial type II cells. We performed RNA sequencing of human alveolar epithelial type II cells transduced with mutant surfactant protein C or control vector.