249There has been a renewed interest in organoid culture in the context of PSC biol-
ogy (Clevers 2016 ; Sasai 2013b). As directed differentiation protocols for an
increasing number of cell lineages of all germ layers become more efficient, organ-
oid culture offers an ideal system to study interactions of multiple PSC-derived cell
types in vitro.
In the field of lung stem cell biology, organoids have been used primarily to
study putative adult stem/progenitor cell populations ex vivo and to establish their
differentiation potential and lineage relationships. Hogan and coworkers used a 3D
system, wherein single mouse trachea or human airway basal cells were embedded
in Matrigel, to assess clonogenicity and multilineage differentiation of these cells
(Rock et al. 2009 ). In both mouse “tracheospheres” and human “bronchospheres,”
single KRT5+ cells self-renewed and gave rise primarily to ciliated cells. The authors
were also able to validate a novel cell surface marker for basal cells, NGFR, in this
system by demonstrating that sorted NGFR+ cells were more clonogenic than
NGFR− cells. In a follow-up paper, the tracheosphere assay was used for screening
of compounds that promote differentiation of mouse trachea basal cells (Tadokoro
et al. 2014 ). IL-6/STAT3 signaling was found to promote ciliated cells and reduce
secretory cell differentiation, and this finding was validated in in vitro human bron-
chial cell differentiation and in vivo mouse models of airway injury. Work from the
same group used “alveolospheres” to demonstrate that type II AECs, or a subset
thereof, are stem cells of the alveolar epithelium since they self-renewed and gave
rise to type I AECs upon single-cell passaging and sphere formation (Barkauskas
et al. 2013 ). Contrary to tracheospheres, alveolosphere formation required coculture
with PDGFRα+ stromal cells, and these cells were also found to be part of the type
II AEC niche in vivo.
Matrigel-induced organoid culture has also been used for the study of putative
bronchioalveolar stem cells (BASCs) (Kim et al. 2005 ; Lee et al. 2014 ) or novel
(β 4 +) (Chapman et al. 2011 ; McQualter et al. 2010 ) stem cell populations or sub-
populations of facultative airway progenitors (Chen et al. 2012 ). In the case of
BASCs, primary mouse lung endothelial cells were able to support BASC prolifera-
tion and differentiation in three different organoid types of distinct morphology and
composition (called bronchiolar, alveolar, and bronchoalveolar) (Lee et al. 2014 ).
The effect of endothelial cells on alveolar differentiation of BASCs was found to be
organ specific (coculture with liver endothelial cells only supported bronchiolar
colonies) and thrombospondin 1 mediated.
Similarly, β 4 + lung cells, currently known as lineage-negative cells (LNEPs)
(Vaughan et al. 2015 ), were extensively characterized in organoid culture (Chapman
et al. 2011 ). Originally, these cells were found to mainly reside within the alveolar
compartment, expressing very low levels of differentiation markers. The authors
reported the development of a novel in vivo organoid assay, in which labeled β 4 +
cells were mixed with embryonic lung epithelial cells and transplanted under the
kidney capsule of immunodeficient mice. The resulting organoids contained distal
(SFTPC+) and proximal (CC10+) areas derived entirely from β 4 + cells, confirming
their multipotency.
13 Development and Bioengineering of Lung Regeneration