Identification of a Proximal Progenitor Population from Murine Fetal Lungs with Clonogenic and Multilineage Differentiation Potential

Abstract

SummaryLung development-associated diseases are major causes of morbidity and lethality in preterm infants and children. Access to the lung progenitor/stem cell populations controlling pulmonary development during embryogenesis and early postnatal years is essential to understand the molecular basis of such diseases. Using a Nkx2-1mCherry reporter mouse, we have identified and captured Nkx2-1-expressing lung progenitor cells from the proximal lung epithelium during fetal development. These cells formed clonal spheres in semisolid culture that could be maintained in vitro and demonstrated self-renewal and expansion capabilities over multiple passages. In-vitro-derived Nkx2-1-expressing clonal spheres differentiated into a polarized epithelium comprised of multiple cell lineages, including basal and secretory cells, that could repopulate decellularized lung scaffolds. Nkx2-1 expression thus defines a fetal lung epithelial progenitor cell population that can be used as a model system to study pulmonary development and associated pediatric diseases.

Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling

“…As we have previously described the use of Wnt3a to drive lung differentiation in murine PSCs (Kurmann et al, 2015; Longmire et al, 2012) (Figure 4A) and because genetic mouse models demonstrate a role for canonical Wnt signaling in proximodistal patterning (Hashimoto et al, 2012; Mucenski et al, 2003; Shu et al, 2005), we differentiated a mouse embryonic stem cell (mESC) line containing an mCherry reporter targeted to the Nkx2-1 locus (Bilodeau et al, 2014; Kurmann et al, 2015) (Figure 4B). Following lung lineage specification into Nkx2-1 + primordial progenitors, on day 14 we replated sorted Nkx2-1 mCherry+ versus Nkx2-1 mCherry− cells (Figure 4C) for further differentiation in sustained versus withdrawn Wnt3a protein.…”

“…Nkx2-1 mCherry mESCs (Bilodeau et al, 2014) were maintained on inactivated mouse embryonic fibroblasts in DMEM supplemented with 15% FBS, L-glut, LIF, and BME (Kurmann et al, 2015; Longmire et al, 2012). Undifferentiated cells were passaged routinely by single cell dissociation using trypsin, enzyme inactivation, and centrifugation at 300xg for 5 min at 4°C and replated onto previously prepared feeder layers on gelatin-coated plastic.…”

Three-Dimensional Culture and FGF Signaling Drive Differentiation of Murine Pluripotent Cells to Distal Lung Epithelial Cells

“…These include the R1 line [27], C2 line [28], iPS EOS3F28 [29], and WT3#6 line ( J. Ellis, unpublished data). An Nkx2-1 mCherry reporter ESC line was used for a subset of experiments to allow for tracking and quantification of Nkx2-1 expression [30].…”

“…To test the efficiency of NKX2-1 induction by early lung mesenchyme, we used an Nkx2-1 mCherry ES cell line to quantify NKX2-1 + progenitors generated in the mesenchyme aggregates [30]. These ESCs were induced as described before and sorted for dual expression of CXCR4 and EPCAM (Fig.…”

Regenerative therapy for hypothyroidism: Mechanisms and possibilities

“…Next, because the Nkx2-1 GFP ESC line likely is haploinsufficient for Nkx2-1, as discussed previously, Kurmann et al compared this line to a novel Nkx2-1 ESC line where a fluorochrome reporter gene (mCherry) had been inserted in the 3’ untranscribed region (UTR) of Nkx2-1 thus allowing for the expression of both endogenous Nkx2-1 alleles (41). While both lines showed similar Nkx2-1 lineage specification efficiencies, the Nkx2-1 mCherry line showed a 2-fold elevation in Nkx2.1 mRNA expression and after sorting and further culturing for 25 days significantly increased expression levels of Pax8, Tg, Tpo, TSHr and NIS.…”