243DE described above, cells are sequentially exposed to factors promoting first ante-
riorization and then ventralization. Anteriorization is promoted by combined inhibi-
tion of the TGF-β and BMP pathways through the application of pathway inhibitors,
such as SB-431542 (pharmacological TGF-β inhibitor) and Noggin (physiological
BMP inhibitor) (Green et al. 2011 ). Together, these inhibitors support the induction
of Sox2 expression, maintenance of Foxa2, and suppression of Cdx2 expression
characteristic of anterior foregut endoderm (AFE). Cells that have undergone dif-
ferentiation to AFE are then exposed to factors known to promote D/V patterning
and separation of the developing esophagus and trachea in vivo. These factors
include agonists of the Wnt pathway (CHIR99021 or WNT3A) together with
BMP4, FGFs (FGF2, FGF7, and/or FGF10), and RA (Chen et al. 2010 ; Green et al.
2011 ; Longmire et al. 2012 ; Mou et al. 2012 ). Differentiating PSCs exposed in cul-
ture to these factors have been shown to have the capacity to subsequently express
Nkx2–1, the earliest known marker of lung epithelium and identifier of cells with the
capacity to further differentiate to the mature parenchymal cells that comprise the
adult lung.
13.5 Patterning and Maturation of Developing Lung
Epithelium
Between E9.5 and E16.5 in the mouse, also referred to as the “pseudoglandular
stage,” the primary lung buds emerge and undergo a stereotyped, iterative branching
process to generate the primary treelike structure of the lung (Metzger et al. 2008 ).
The primary buds extend into the surrounding mesenchyme, and by E10.5, second-
ary buds appear at specific locations around the primary bud. This process, known
as branching morphogenesis, continues for the remainder of the pseudoglandular
stage of lung development, ultimately giving rise to the main stem bronchi and the
entire bronchial tree. The branching morphogenesis program is governed by the
establishment of a signaling center at the distal lung tip in which FGF10 is expressed
from the mesenchyme and, together with BMPs and Wnts, signals to the adjacent
epithelium, maintaining proliferation of SOX9+/ID2+ epithelial cells. Reciprocal
signaling from the epithelium, meanwhile, including expression of Sprouty, Shh,
and BMP4, restricts FGF expression and establishes a feedback loop that controls
the size of the progenitor pool as well as the size and shape of the lung bud (Cardoso
and Lu 2006 ). As epithelial cells of the distal bud proliferate, the bud itself elongates
resulting in the formation of a stalk that experiences different expression levels of
the key factors listed above (see reviews by Cardoso and Lu 2006 ; Morrisey and
Hogan 2010 ). A repetitive cycle of bud elongation, tip expansion, and bifurcation
occurs during this time, during which relatively proximal and distal cells experience
varying levels of key morphogens. Components of the Hh pathway play a central
role in this process: epithelial Shh promotes proliferation of adjacent mesenchymal
cells and expression of Bmp4 while at the same time inhibiting Fgf10 expression.
13 Development and Bioengineering of Lung Regeneration