Human lung development: recent progress and new challenges

Abstract

Recent studies have revealed biologically significant differences between human and mouse lung development, and have reported new in vitro systems that allow experimental manipulation of human lung models. At the same time, emerging clinical data suggest that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. In this Review, we discuss our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments using in vitro human lung developmental models and RNA sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on the mechanisms underlying human lung development and disease, and may help pave the way for new therapies.

Keywords: Alveolar; Bronchi; ESC; Lung disease; Progenitor; Stem cell; iPSC.

Fig. 1.

Human adult lung structure and cell types. Lobular structure of the human adult lung. Insets depict the cell types found within the airway epithelium (left) and the alveolar epithelium (right).

Fig. 2.

Stages of human lung development. (A) Schematics depicting general lung morphology across the five different stages of human lung development: embryonic, pseudoglandular, canalicular, saccular and alveolar. For each stage, the developmental period is indicated, for human in post-conception weeks (pcw) and for mouse in embryonic days (E) and postnatal days (P). Boxed area is enlarged to show the gas exchange occurring across the alveolar epithelium. (B,B′) Cryosection of an embryonic stage lung showing the primary branches and SOX2/SOX9 co-expression in the tips; the boxed area is magnified in B′. (C,D) Cryosections of pseudoglandular stage lungs showing ongoing tip SOX2/SOX9 co-expression and airway differentiation as indicated by the expression of smooth muscle actin (SMA) (C, white), which marks smooth muscle cells, and TP63 (D, green), which marks differentiating basal cells. (E,F) Cryosections of canalicular stage lungs showing SOX9⁺/SOX2⁻ distal tips (in E). Alveolar differentiation is initiated at this stage, as indicated by the widening alveolar spaces. Proximity to developing vasculature, as marked by VE-cadherin (VECAD; CDH5) (green) and podoplanin (PDPN, red) is illustrated in F. Note that there is a gap in the available images at the saccular stage where distal tips are presumed to disappear and alveolar differentiation progresses. (G,H) Cryosections of alveolar stage postnatal lungs, showing the expression of SOX9 (cartilage, green), SOX2 (airway cells, red) and ACTA2 (smooth muscle, white) in G, and NKX2-1 (lung epithelium, green), FOXF1 (mesenchyme) and ACTA2 (smooth muscle, white) in H. At this stage, SOX9⁺ distal tips are no longer seen (G), but there has been continued growth and septal formation to make alveoli (H). Images in A-F are reproduced from Nikolić et al., 2017. Images in G and F were kindly provided by Jeff Whitsett, University of Cincinnati College of Medicine (https://research.cchmc.org/lungimage/). Scale bars: 200 μm (B); 50 μm (C,D,F); 100 μm (E,G,H).

Fig. 3.

Selected differences between mouse and human lung development. (A) Comparison of human and mouse distal epithelial tip transcriptomes reveals shared and unique transcripts (Nikolić et al., 2017). (B) One specific example of a molecular difference between mouse and human lungs is that SOX2 expression extends to the distal epithelial tip in pseudoglandular stage human lungs, but not in mouse. However, by the canalicular stage, human and mouse lungs have similar SOX2 expression in the differentiating airway only. (C) Mouse (Nichane et al., 2017) and human (Miller et al., 2017; Nikolić et al., 2017) distal epithelial tip progenitors are maintained in culture via the activation, or inhibition, of different signalling pathways. Asterisks indicates factors reported by Miller et al., 2017 that were needed in addition to those reported by Nikolić et al., 2017 for the self-renewal of human epithelial distal tip progenitors.

Fig. 4.

Methods for ex vivo culture of developing human lung tissue. (A) Culture set-up for distal tip organoids. Whole distal epithelial tips are placed within Matrigel and growth medium is added on top. (B-D) Other available culture systems for human developing lungs and cells include immune-compromised mouse kidney capsule grafting (B), seeding cells on alginate beads in bioreactors (C), culture of lung explants floating at the surface (or submerged within) growth medium (D) and co-culture with matrix-embedded fibroblasts (E).

Fig. 5.

Studying human lung development using hPSCs. The usual strategy for differentiating human pluripotent stem cells (hPSCs) to lung cells relies on maturing the cells through sequential progenitor stages that correspond, as closely as possible, to normal embryonic development. Typically, hPSCs, which are equivalent to the cells found in the inner cell mass of the blastocyst (ICM, day 6 post-conception), are differentiated to SOX17⁺/FOXA2⁺ definitive endoderm (∼3 pcw, equivalent to E7.5 in mouse). These cells are then matured to SOX2⁺/FOXA2⁺ anterior foregut endoderm (∼4 pcw, equivalent to E8.5-E9.5 in mouse) from which the lungs bud. Further differentiation proceeds via a NKX2-1⁺/SOX2⁺ ventralised anterior foregut endoderm that corresponds to the NKX2-1⁺ lung foregut progenitor domain characterised in mice (5-6 pcw). NKX2-1 is known to be also expressed in the developing brain and thyroid, hence TUJ1⁻/PAX8⁻ marker selection is also used to define NKX2-1⁺ lung progenitor cells in vitro. Current hPSC stepwise differentiation protocols sort a pure population of NKX2-1⁺/SOX2⁺/TUJ1⁻/PAX8⁻ lung progenitor cells, which are subsequently differentiated towards airway or alveolar fate by modulation of Wnt signalling. Key signalling pathways are depicted with a more detailed summary in Table S1. A, anterior; D, dorsal; DP, dorsal pancreas; Int, intestine; L, lung; Li, liver; P, posterior; RA, retinoic acid; St, stomach; V, ventral; VP, ventral pancreas.