248
et al. 2011 ) can be rigorously tested by characterizing the spatial distribution and
organization of proximal and distal cell types derived from purified lung progenitors
on lung slices and eventually in whole decellularized lungs.
A recent report has partially addressed these questions by using lung embryonic
progenitors that were purified using an Nkx2–1mCherry reporter mouse (Bilodeau
et al. 2014 ). The authors recellularized rat lung scaffolds with either freshly isolated
or clonally expanded E12.5–E14.5 proximal lung epithelial progenitors. In both
cases, the progenitors gave rise to organized and polarized pulmonary epithelia that
contain several proximal (SOX2+) cell types, such as secretory, ciliated, and basal
cells. There was absent expression of the distal markers SFTPC and PDPN1 even in
distal areas of the scaffold, indicating that distalizing ECM signals cannot instruct
the differentiation of proximal lung progenitors.
Another type of decellularized scaffold, denuded trachea scaffolds, has been pre-
viously used in lung epithelial biology to test the differentiation and proliferation
potential of tracheal epithelial cells such as club and basal cells (Hook et al. 1987 ;
Inayama et al. 1988 ; Randell et al. 1991 ). In this application, rat tracheas are decel-
lularized by repeated freeze-thawing, recellularized by epithelial tracheal cells, and
transplanted subcutaneously into nude mice. Although there are currently no reports
on its use in combination with PSC-derived cells, it will undoubtedly become a
platform of choice for the functional characterization of PSC-derived proximal lung
progenitors and downstream lineages.
As lung-directed differentiation protocols become more refined leading to the
efficient derivation of diverse proximal and distal cell types, decellularized lung or
tracheal scaffolds will remain an essential tool for the elucidation of the effect of
biomechanical signals, including stiffness and ECM, on lung differentiation and
maturation.
13.8 3D Lung Organoid Culture
Ex vivo organoids, i.e., multilineage cell assemblies that recapitulate key morpho-
logical and functional attributes of an organ, have been used in developmental biol-
ogy for more than 100 years (Lancaster and Knoblich 2014 ). In vitro or ex vivo
organoid formation is based on and takes advantage of the universal biological prin-
ciples of self-assembly, self-organization, and self-patterning through mechanisms
of cell commitment, proliferation, migration, and differential adhesion (Lancaster
et al. 2013 ; Sasai 2013a, b). For example, when E16.5 lungs were monodispersed
and cells were placed on filters in air-liquid interface culture, emergence of two
distinct structures that later gave rise to differentiated lung epithelium and connec-
tive tissue was observed (Zimmermann 1987 ). In a similar vein, lung mesenchyme
recombinants have been used extensively for the ex vivo study of lung epithelial-
mesenchymal interactions and developmental competence of early proximal and
distal lung epithelium (Shannon 1994 ; Shannon et al. 1999 ; Shannon and Hyatt
2004 ).
A. Wilson and L. Ikonomou