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-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 is operative in mouse primary keratinocytes in conventional cultures as determined by the nuclear Smad2/3 localization. Accordingly, TGF-β 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-β 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.
Highlights
Homeostasis and regeneration of epithelia are maintained by self-renewal, proliferation, and differentiation of tissue-specific stem cells[1,2,3]
We show that the use of a single small molecule inhibitor of Transforming growth factor-β (TGF-β) signaling in crude mouse tissue preparations enriches p63+ epithelial progenitor cells and supports their long-term proliferative capacity in a low Ca2+ condition
We describe a simple and effective method of expanding p63+ epithelial progenitor cells from crude tissue preparations of diverse mouse epithelia using a single small molecule inhibitor of TGF-β signaling in a low Ca2+, feeder-free condition
Summary
Inhibition of TGF-β signaling stimulates the proliferation of mouse primary epidermal cells in vitro. We show that p63+ epidermal progenitor cells derived from 4-week-old mice grew at a constant rate for at least 60 days in a RepSox-dependent manner (Fig. 4h) Together, these results indicate that inhibition of TGF-β signaling in crude primary cell culture of postnatal mouse epidermis selectively expands p63+ epidermal progenitor cells and supports their proliferative potential long term. Crude preparations of primary cultures of all p63-dependent epithelia contained non-epithelial cells and terminally differentiated p63− epithelial cells, just as in epidermis, >90% of cells from all epithelial cultures showed high p63 expression by P1 to P2 (Figs 5b and S6b), and virtually all growing cells were p63+ thereafter in all cultures derived from both newborn and 4-week-old mice These results indicate that inhibition of TGF-β signaling in CnT-PR media selectively expands p63+ epithelial progenitor cells in crude tissue preparations of diverse mouse epithelia. The mechanisms of how cFAD induces differentiation of each epithelial cell type need to be determined, these results indicate that p63-dependent mouse epithelial progenitor cells expanded by TGF-β signaling inhibition are capable of differentiation in vitro
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