247(Daly et al. 2012 ; Wallis et al. 2012 ). In this technology, decellularized lungs (most
commonly of murine origin) are recellularized using a mixture of cells and agarose,
sliced to thick pieces (300–2000 μm) and placed into culture media where agarose
melts out of the tissue (Daly et al. 2012 ). Decellularized lung slices retain the origi-
nal architecture of the airway, alveolar, and endothelial compartments thus offering
an expedient and inexpensive system for functional studies of PSC-derived lineages
(Gilpin et al. 2014b; Longmire et al. 2012 ; Shojaie et al. 2015 ). In the first study
reporting the efficient derivation of lung/thyroid lineages from mouse ESCs, puri-
fied mouse NKX2–1+ endodermal progenitors retained NKX2–1 expression in
mouse lung slice culture giving also rise to NKX2–1−T1α+ cells, a phenotype remi-
niscent of type I AECs (Longmire et al. 2012 ). Niklason and coworkers described
similar results with presumed type II AECs derived from human iPSCs (Ghaedi
et al. 2013 ). Recellularization of whole lung scaffolds and rat/human lung slices
resulted in proliferating cells that expressed NKX2–1 and markers of distal lung
epithelium.
The potential of this culture system to enhance lung epithelial progenitor speci-
fication from PSCs was evaluated in two studies (Gilpin et al. 2014b; Shojaie et al.
2015 ). Ott and coworkers (Gilpin et al. 2014b) reported enhanced lung specification
of human iPSCs during directed differentiation on lung slices compared to non-slice
controls. Specified progenitors were further differentiated to cells expressing mark-
ers of proximal (CC10) and distal lung lineages (T1α), and the recellularized scaf-
folds were able to support lung function posttransplantation into pneumonectomized
rats. Shojaie and colleagues (Shojaie et al. 2015 ) investigated the inductive proper-
ties of lung ECM alone by seeding acellular lung scaffolds with PSC-derived DE
cells in growth factor-free media. Intriguingly, about 45% of the cells expressed
NKX2–1 after 3 weeks in culture, and these cells seemed to adopt predominantly
proximal fates as demonstrated by staining for basal, secretory, ciliated, and mucin-
producing cell markers. This report along with an older study that claimed a major
contribution of ECM cues to lung differentiation of mouse ESCs (Cortiella et al.
2010 ) appears to contradict a growing body of literature that has demonstrated deri-
vation of AFE via inhibition of TGF-β/BMP signaling and subsequent use of solu-
ble agonists of BMP and Wnt signaling to be prerequisites of murine and human
PSC lung specification (Gotoh et al. 2014 ; Huang et al. 2014 ; Longmire et al. 2012 ;
Mou et al. 2012 ; Rankin et al. 2016 ). Studies that involve manipulation of TGF-β/
BMP/Wnt signals in lung scaffold culture of PSC-derived endodermal cells will be
required to settle this controversy. Taken collectively, the abovementioned studies
do point to an important role of native lung ECM in lung-directed differentiation of
PSCs. Therefore, lung slice culture can be used to systematically test formal hypoth-
eses regarding ECM signaling functions in lung-directed differentiation. For exam-
ple, the tissue specificity of the observed lung ECM effects can be systematically
addressed by seeding of purified PSC-derived multipotent lung progenitors on tis-
sue slices, micro-scaffolds (Shamis et al. 2011 ), or solubilized matrices from vari-
ous decellularized organs (e.g., lung, liver, heart, brain) and detailed molecular and
phenotypic characterization of the resultant differentiated lineages. Similarly, the
hypothesis of ECM “zip codes” for cell attachment and differentiation (Badylak
13 Development and Bioengineering of Lung Regeneration