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The high levels of Shh expression at the distal lung bud tip induce expression of
hedgehog-interacting protein (Hhip) in the adjacent mesenchyme that dampens Gli
signaling and thereby allow expression of Fgf10. More proximal regions experience
lower Shh expression that, by contrast, does not induce Hhip resulting in Fgf10
inhibition. Epithelial cells that become displaced from the distal tip form stalk
regions of the airways and adopt a SOX2+ proximal epithelial fate. Additional fac-
tors not detailed above, including TGF-β signaling, Wnt7b/β-catenin signaling, and
Fgf9, likewise play important roles in this process.
A limited understanding of the dynamic epithelial-mesenchymal signaling inter-
actions that occur during the pseudoglandular period together with associated spa-
tial complexity has made the reproduction of this stage of lung development
challenging to recapitulate in vitro. A key step in the ability to derive differentiated
lung cell types from PSCs is the establishment of the proximal (Nkx2–1+/Sox2+) or
distal (Nkx2–1+/Sox9+) cellular identity that will ultimately produce airway or alve-
olar epithelial cells (AECs), respectively. While the precise cues required to direct
Nkx2–1+ lung progenitors to adopt a proximal or distal identity have not been estab-
lished, specific protocols have demonstrated the ability to generate cells expressing
markers of differentiated lung cell types. To generate putative proximal lung epithe-
lial cells, several protocols have relied on physical manipulation of the culture envi-
ronment to assist in cellular maturation. For example, one publication demonstrated
the use of subcutaneous injection and subsequent culture of mouse or human pro-
genitor cells to induce a variety of differentiated markers, including P63, CC10, or
MUC5AC (Mou et al. 2012 ). Multiple reports have now demonstrated the use of
air-liquid interface culture to further induce expression of differentiated cell mark-
ers and/or ciliated cells following directed differentiation of human PSCs to gener-
ate putative lung progenitors (Firth et al. 2014 ; Wong et al. 2012 ). Most recently,
Konishi et al. sorted cells expressing the surface marker carboxypeptidase M (CPM)
to identify and purify Nkx2–1-expressing cells from ventralized AFE cultures
(Konishi et al. 2016 ). CPM+ cells were then cultured in 3D conditions in the pres-
ence of the WNT agonist CHIR99021 and FGF10 to generate 3D spherical struc-
tures before further culture in a commercial bronchial airway cell culture medium
together with DAPT, an inhibitor of NOTCH signaling. These culture conditions
generated FOXJ1+ motile multiciliated cells as well as cells expressing the neuroen-
docrine markers CHGA and SYP. Numerous reports have likewise demonstrated the
ability to derive lung epithelial cells capable of expressing the type II AEC marker
SFTPC from either mouse or human PSCs (Ghaedi et al. 2013 ; Green et al. 2011 ;
Huang et al. 2014 ; Longmire et al. 2012 ). Protocols to induce expression of this key
marker feature the induction of Nkx2–1+ progenitor cells then cultured in the
presence of growth factors including Wnt agonists, FGF2 and/or FGF10, KGF,
BMP4, EGF, and RA. Increased expressions of SFTPC and SFTPB were achieved
in these publications through subsequent culture in conditions including KGF and/
or dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthine (together referred
to as “DCI”) (Huang et al. 2014 ; Longmire et al. 2012 ).
A. Wilson and L. Ikonomou