Basal cells as stem cells of the mouse trachea and human airway epithelium

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2009; 106(31):12771-5. DOI: 10.1073/pnas.0906850106
Source: PubMed


The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER(T2) transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.

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    • "This basal cell population, both in mice and human, have extensive proliferative potential, self-renewal capacity and ability to differentiate into secretory club and ciliated cells[102,104]. The 3D " tracheosphere " culture of FACS-sorted pure population of Krt5 + p63 + NGFR + basal cells shows clonal expansion of differentiated cells which are positive for both ciliated and secretory club (Clara) cells[102,103]. "
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    ABSTRACT: The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.
    Full-text · Article · Feb 2016 · International Journal of Molecular Sciences
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    • "Ngfr+ BCs isolated as described (Rock et al., 2009) from C57Bl/6 or Foxj1- GFP mice were suspended in MTEC/plus medium, mixed 1:1 with growth factorreduced Matrigel (Corning Life Sciences), and seeded at 1,000 cells/well in 24 well 0.4 um pore inserts (#3470, Corning Life Sciences). Factors were added to the medium in the lower well, and medium changed every other day. "
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    ABSTRACT: The pseudostratified epithelium of the lung contains ciliated and secretory luminal cells and basal stem/progenitor cells. To identify signals controlling basal cell behavior we screened factors that alter their self-renewal and differentiation in a clonal organoid (tracheosphere) assay. This revealed that inhibitors of the canonical BMP signaling pathway promote proliferation but do not affect lineage choice, while exogenous BMP4 inhibits proliferation and differentiation. We therefore followed changes in BMP pathway components in vivo in the mouse trachea during epithelial regeneration from basal cells after injury. The findings suggest that BMP signaling normally constrains proliferation at steady state and this is break is released transiently during repair by the upregulation of endogenous BMP antagonists. Early in repair the packing of epithelial cells along the basal lamina increases, but density is later restored by active extrusion of apoptotic cells. Systemic administration of the BMP antagonist LDN-193189 during repair initially increases epithelial cell number but, following the shedding phase, normal density is restored. Taken together, these results reveal critical roles for both BMP signaling and cell shedding in homeostasis of the respiratory epithelium.
    Preview · Article · Jan 2016 · Development
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    • "Cell-surface antigens including CD44, MUC1, and nerve growth factor receptor (NGFR) and intracellular antigens CK19, p63, CK13, and CK4 were all expressed, but variably , in the surface epithelium, where their location could be associated with an increasingly differentiated state from the basal layer to the outer surface. For example, CK19 and NGFR were exclusive to the basal layer of the surface epithelium (Figure 1I, center row), whereas p63, a widely annotated marker of basal epithelial cells (Pellegrini et al., 2001; Rock et al., 2009; Senoo et al., 2007), and CD44, a receptor for hyaluronic acid, were seen not only in the basal layers but also in the parabasal layers, extending up to the mid-layers of the surface epithelium (Figure 1I, center row). CK13, CK4, involucrin , and cell-surface MUC1 were detected only in the parabasal and upper layers of the surface epithelium and not in any basal cells (Figure 1J, center row). "
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    ABSTRACT: Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44 + NGFR + and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44 + NGFR + cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44 + NGFR + cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcrip-tional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation.
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