Directing lung endoderm differentiation in pluripotent stem cells

Abstract

The lung is composed of numerous epithelial lineages that arise from the anterior foregut endoderm. This review discusses how insights into the signaling mechanisms that regulate lung endoderm specification and subsequent differentiation have recently been exploited to direct differentiation of hESCs/iPSCs into expandable lung progenitors.

Figure 1. Signaling pathways that direct lung endoderm development

The foregut endoderm gives rise to the lung epithelium. FGF signals from the cardiac mesoderm pattern the foregut endoderm into organ specific fields. High levels of FGF signaling promote lung and thyroid specification, and lower levels promote liver specification. BMP and Wnt signals from the splanchic mesoderm promote expression of Nkx2.1 in the ventral endoderm. At E9.0 the trachea bifurcates from the foregut endoderm and the primary lung buds form. FGF10 signals from the mesenchyme drive lung bud outgrowth and is in turn regulated by retinoic acid repression of TGF-B signaling. By E12.5 the 5 lobes of the lung have formed, and stereotyped branching morphogenesis has begun. Proximal progenitor cells express Sox2 and will give rise to the cell types that populate the upper airways (Clara cells, ciliated cells, neuroendocrine cells). Distal progenitor cells express Sox9 and Id2. Early in development (prior to E13.5) these distal cell progenitors can give rise to all the epithelial cells of the lung. Later in development (after E16.5), these distal progenitors will generate the alveolar epithelial lineages including AEC1 and AEC2 cells. In the adult lung (bottom panel) the upper airways are populated with Clara Cells (CC10+), ciliated cells (Foxj1+), basal cells (p63+) and neuroendocrine cells. The mature alveolus is the site of gas exchange in the lung, lined with AEC1 and AEC2 cells. AEC1 cells have flattened morphology, express aquaporin5 and T1α, and form close associations with the underlying vasculature. AEC2 cells have a more cuboidal shape and express surfactant protein C (Sftpc).

Figure 2. Recapitulation of the steps of lung endoderm development in vitro promotes lung cell derivation from pluripotent stem cells

Treatment of ESCs or iPSCs with Activin A mimics Nodal signaling required for formation of the primitive streak and formation of mesoendoderm. Continued exposure to Activin A promotes formation of the definitive endoderm. Inhibition of BMP signaling and TGF-β signaling promotes expression of anterior foregut markers, Sox2 and FoxA2. Treatment of the anteriorized endoderm with Wnt, FGF, EGF, KGF and BMP signaling promotes formation of ventral anterior foregut cells that express Nkx2.1. Treatment with FGF2 or FGF10 ligands promotes specification of lung progenitors, which express Nkx2.1, Sox2 and Sox9. Further maturation of the lung progenitors can be induced by treatment with a ‘proximal induction’ cocktail (Mou et al., 2012) or lung maturation media (Longmire et al., 2012). Injection of cells into decellularized lung scaffold or subcutaneously promotes further differentiation into proximal and distal cell types.