silybin The relationships of the proximal fetal lung progeni
The relationships of the proximal fetal lung progenitor pool characterized in this study to other epithelial cell types remain to be investigated. In the proximal lung of adult animals, either club silybin expressing SCGB1A1 or basal cells expressing KRT5, ITGA6, and NGFR (and others) acts as multipotent progenitor/stem cells (Rawlins et al., 2009b; Rock et al., 2009; Tata et al., 2013). However, at the stage of fetal development that we studied (i.e., E12.5–E14.5), the mature cells characterized by these markers are not detectable yet. These early proximal progenitors are also distinct from other populations previously characterized in murine fetal lungs. For example, they functionally differ from early secretory cells expressing Scgb3a2 and Upk3a (Guha et al., 2012) because they give rise to basal and submucosal gland cells in addition to club and ciliated cells. Also, their proximal location and lineage contribution differ from the distal multipotent tip cells or bipotent alveolar progenitors (Desai et al., 2014; Rawlins et al., 2009a). Therefore, we believe that our characterization of early proximal lung progenitors extends our knowledge of the cellular mechanisms of fetal pulmonary development. Postnatal basal cells are multipotent stem cells in the mouse and human upper airways and, perhaps, in the human lower airways where they are also found (Rock et al., 2009). Prebasal cells expressing P63 were found as early as E10.5 in mouse proximal lungs. Fractionation of E14.5 mC+ cells with ITGB4 revealed that the colony-formation frequency did not correlate with the enrichment of prebasal cells in the ITGB4+Hi fraction. Therefore, our results suggest either that nonbasal epithelial cells adopt a basal-progenitor fate when isolated from other cells, as observed with adult lungs (Tata et al., 2013), or a nonbasal or undifferentiated cell population(s) of the fetal trachea is endowed with in vitro clonogenic self-renewal and differentiation potential. Supporting the latter possibility, a tracheal epithelium comprised of ciliated and other columnar cells does develop in fetal mice lacking basal cells (Daniely et al., 2004), and we efficiently derived more colonies at E12.5 when P63+ cell numbers were lower than at E14.5. Therefore, additional clonal and functional analyses will be required to distinguish whether the progenitor activity is a property of a specific cell subpopulation(s) or whether all early Nkx2-1+ cells act as facultative progenitors depending on the environmental context. Our findings are likely relevant to human biology. In human lungs, basal cells are detectable at gestational age 25 weeks and later (KRT14+) (Broers et al., 1989). Up to gestational age 10 weeks, the lung epithelium comprises unspecialized columnar cells (Broers et al., 1989). Undifferentiated fetal human lung bud tissues or cell suspensions (stage 5–8 weeks) can reconstitute a pseudostratified mucociliary epithelium in vivo in denuded trachea explants (Delplanque et al., 2000). In comparison to our study, 8-week-old fetal human lungs correlate with E12.0 murine lungs (Kimura and Deutsch, 2007). Here, we show that some E12.5 mouse cells isolated from the poorly differentiated proximal airway epithelium are multipotent and self-renewing. Identification of this same progenitor population during differentiation of human pluripotent stem cells to lung cells could provide a renewable resource of lung progenitors for disease modeling and lung repair. Finally, our Nkx2-1mCherry reporter mouse line, phenotypically normal in a homozygous state, is a practical alternative to the Nkx2-1EGFP reporter mouse previously reported, which has to be maintained in a heterozygous state because one allele of the gene is disrupted (Longmire et al., 2012). Our mouse line is advantageous for in vivo experiments because heterozygous or homozygous Nkx2-1 mutations are haploinsufficient or lethal, respectively (Kimura et al., 1996; Pohlenz et al., 2002).