In the adult mouse and
In the adult mouse and human lungs, distinct region-specific epithelial progenitor cells have been described (Wansleeben et al., 2013), but their fetal counterparts remain undercharacterized. At E10.5–E12.5, Id2-expressing distal tip cells of the fetal lung buds are multipotent and contribute to the conducting airways (e.g., club, ciliated, neuroendocrine) and alveolar (AT1 and AT2) lineages (Rawlins et al., 2009a). Later at stage E15.0, AT1 and AT2 cells derive from a bipotent progenitor (Desai et al., 2014). Inducible lineage tracing regulated by the Cgrp promoter (neuroendocrine cell marker) at E12.5–E14.5 labels neuroendocrine and alveolar (AT1 and AT2 cells) descendants (Song et al., 2012). However, Ascl1-expressing cells are reported to give rise to airway, AT2, and nonepithelial cells, a finding to be clarified by clonal analysis (Li and Linnoila, 2012). Also, secretory cells contribute to club and ciliated lineages postnatally (Guha et al., 2012). Although these studies reveal the origin of intrapulmonary airways and silybin lineages (i.e., distal lung), the progenitor relationships in the proximal trachea and extrapulmonary bronchi (i.e., proximal lung) remain mostly unresolved. Inducible lineage tracing driven by the human SPC promoter suggests a distinct origin for proximal and distal lungs (Perl et al., 2002). Moreover, fetal human tracheal tissue can mature into basal, mucociliary, and submucosal gland cells after serial xenotransplantation, suggesting progenitor/stem cell activity (Delplanque et al., 2000).
To better understand lineage relationships in fetal lung development, we knocked an mCherry reporter gene into the Nkx2-1 locus to isolate purified primary lung epithelial cells that we submitted to in vitro clonogenic progenitor assays. NKX2-1 is the earliest marker of pulmonary fate and is broadly expressed in the proximal and distal fetal lung epithelium (Kimura and Deutsch, 2007). Nkx2-1-deficient mice are stillborn and show severe distal lung epithelium branching and cytodifferentiation defects (Kimura et al., 1996; Minoo et al., 1999). Also, their trachea epithelium fails to separate from the esophagus and adopts an esophagus-like phenotype, with high expression of SOX2 and P63 (Minoo et al., 1999; Que et al., 2007). Our molecular characterization of Nkx2-1-expressing cells reveals an underappreciated broad cellular diversity in the airways, including progenitor cells with long-term clonogenic and differentiation potential in vitro. These cells self-renew and engraft when seeded onto decellularized lung scaffolds. Overall, these results suggest that the Nkx2-1-expressing population in the fetal proximal airways contains cells that can act as self-renewing multilineage progenitors in vitro.
Discussion In this study, we demonstrated that a subset of Nkx2-1-expressing cells, derived from the mouse fetal proximal airways, has in vitro clonogenic and multilineage differentiation potential toward airway surface epithelium and submucosal gland cells. Nkx2-1-expressing cells from E12.5–E14.5 lungs could generate single-cell-derived epithelial spheres that could be propagated and passaged for an extended period of time in culture due to their self-renewal capacity. These spheres contained differentiated cells predominantly expressing markers of basal and secretory lineages, with less efficient generation of ciliated and submucosal gland cells. Our findings are in agreement with published work suggesting that the submucosal glands are derived from the tracheal epithelium postnatally (Engelhardt et al., 1995). Moreover, our data extend the observations that the early lung anlage has the potential to generate both tissues (Péault et al., 1994) and suggest that the tissues arise from clonogenic multipotent progenitors already present in the proximal region at stage E14.5. An additional feature of our system compared to other in vitro methods to maintain clonal adult proximal progenitors/stem cells (Hegab et al., 2011; Rock et al., 2009) is the enhanced ability to capture precursors that will differentiate into mature secretory club cells. Not only could we readily detect SCGB1A1 expression by immunostaining, but also ultrastructural analysis revealed the presence of cells covered with microvilli and actively producing secretory granules. Moreover, our culture conditions allow the obtainment of a diversity of club cells according to the expression of region-specific markers such as Reg3g, Gabrp, and Upk3a (Guha et al., 2014).