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Inhibition of TGF-? signaling supports high proliferative potential of diverse p63+ mouse epithelial progenitor cells in vitro.

ABSTRACT: Mouse models have been used to provide primary cells to study physiology and pathogenesis of epithelia. However, highly efficient simple approaches to propagate mouse primary epithelial cells remain challenging. Here, we show that pharmacological inhibition of TGF-? signaling enables long-term expansion of p63+ epithelial progenitor cells in low Ca2+ media without the need of progenitor cell-purification steps or support by a feeder cell layer. We find that TGF-? signaling is operative in mouse primary keratinocytes in conventional cultures as determined by the nuclear Smad2/3 localization. Accordingly, TGF-? signaling inhibition in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells, while preserving their ability of differentiation in response to Ca2+ stimulation. Notably, inhibition of TGF-? signaling also enriches and expands other p63+ epithelial progenitor cells in primary crude cultures of multiple epithelia, including the cornea, oral and lingual epithelia, salivary gland, esophagus, thymus, and bladder. We anticipate that this simple and efficient approach will facilitate the use of mouse models for studying a wide range of epithelia by providing highly enriched populations of diverse p63+ epithelial progenitor cells in quantity.

SUBMITTER: Suzuki D

PROVIDER: S-EPMC5519764 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Inhibition of TGF-β signaling supports high proliferative potential of diverse p63+ mouse epithelial progenitor cells in vitro.

Suzuki Daisuke D Pinto Filipa F Senoo Makoto M

Scientific reports 20170720 1

Mouse models have been used to provide primary cells to study physiology and pathogenesis of epithelia. However, highly efficient simple approaches to propagate mouse primary epithelial cells remain challenging. Here, we show that pharmacological inhibition of TGF-β signaling enables long-term expansion of p63+ epithelial progenitor cells in low Ca2+ media without the need of progenitor cell-purification steps or support by a feeder cell layer. We find that TGF-β signaling ...[more]

PMID: 28729719

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Project description:Background: The Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs). METHODS: HMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs. RESULTS: EpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27KIP1 and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-beta1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes. CONCLUSIONS: EpCAM revealed oncogenic features in normal human breast cells by, inducing resistance to TGF-beta1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands. For each gene a representative transcript probe set was selected based on a score that combines average expression and variation of expression across all samples. Transcript probe sets with more than 4 probes were preferred. Differential expression analysis between EPCAM over-expressing and control cells (expressing GFP) was performed using a paired moderated t-test employing functions provided by Bioconductor's limma package. p-values were subsequently adjusted for multiple hypothesis testing using the method from Benjamini and Hochberg for a strong control of the false discovery rate.

Dataset Information

Inhibition of TGF-? signaling supports high proliferative potential of diverse p63+ mouse epithelial progenitor cells in vitro.

Publications

Inhibition of TGF-β signaling supports high proliferative potential of diverse p63<sup>+</sup> mouse epithelial progenitor cells in vitro.

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