239current time. While the ability to efficiently derive certain cell types and tissues,
such as hepatic or neuronal cells, in vitro from PSCs has been well established,
protocols to achieve this goal for lung-derived cells have only recently and incom-
pletely been developed (Green et al. 2011 ; Huang et al. 2014 ; Konishi et al. 2016 ;
Longmire et al. 2012 ). Progress in this approach has largely resulted from the cul-
ture of PSCs and their differentiated progeny in conditions designed to mimic those
encountered by their in vivo analogues during development. In this section, we will,
therefore, review what is known about each stage of lung development in the devel-
oping embryo and how this developmental road map is then applied to the in vitro
differentiation of PSCs.
13.3 Derivation of the Endodermal Germ Layer
The lung, together with the thyroid, liver, intestines, and pancreas, is derived from
the endodermal germ layer. Definitive endoderm is formed during gastrulation, a
process that begins at embryonic day (E) 6.5 in the mouse (Tam et al. 2006 ). During
this process, a portion of the epiblast forms a structure termed the “primitive streak.”
Additional epiblast cells migrate through the primitive streak, exiting the streak in
an ordered fashion and in the process giving rise to mesodermal or endodermal cell
types based on their specific embryonic location. This process, known as gastrula-
tion, occurs from E7.5 to E8.5 in the mouse and results from the temporospatial
expression of key growth factors and inhibitors that establish signaling gradients in
the epiblast. Most notably, gradients of the TGF-β, Nodal, and Wnt ligands signal
across the anterior/posterior (A/P) axis, while a BMP4 gradient is established along
the proximal-distal axis (Fig. 13.1) (Gadue et al. 2005 ). Cells migrating through the
streak experience variable and fate-altering levels of Nodal based on this A/P gradi-
ent, with posteriorly emerging cells becoming mesoderm while anteriorly emerging
cells form definitive endoderm (DE) (Zorn and Wells 2009 ). Wnt signaling through
β-catenin, meanwhile, helps to maintain Nodal signaling to promote endoderm
induction and together with Smad2 promotes the expression of key endodermal
genes such as Foxa2 and Sox17 (Sinner et al. 2004 ; Zorn and Wells 2009 ). Post-
gastrulation (approximately E8.5–E9.0), DE then forms the primitive gut tube that
is patterned into foregut, midgut, and hindgut regions, each marked by expression
of specific transcription factors, along its A/P axis (Cardoso and Lu 2006 ; Wells and
Melton 1999 ). Reciprocal signaling between the endoderm and mesoderm helps to
confer regional fate specification along the A/P axis, with secretion of retinoic acid
(RA), Wnts, FGFs, and BMPs by posterior mesoderm inducing intestinal identity,
while Wnt and BMP antagonists released anteriorly block these posteriorizing sig-
nals in endodermal cells that will ultimately give rise to the lung, liver, and
pancreas.
The exposure of PSCs in vitro to key components of these essential signaling
pathways can result in the efficient induction of DE, the germ layer that will ulti-
mately give rise to lung epithelium. As during migration of epiblast cells through
13 Development and Bioengineering of Lung Regeneration