201the duration of CHIR99021-mediated specification between 2 and 5 days before the
addition of FGF9 (Takasato et al. 2015 ). A shorter duration of canonical Wnt induc-
tion, particularly with the addition of RA, patterned posterior PS and then anterior
IM, as evidenced by the formation of a GATA3+ epithelium representative of neph-
ric duct, whereas prolonged Wnt signalling, particularly in the presence of an inhib-
itor of RA signalling, increased the expression of the MM transcription factor,
HOXD11, suggesting more posterior IM (Takasato et al. 2015 ). Of note, an interme-
diate duration of canonical induction prior to FGF9 generated cultures within which
there was evidence for both anterior and posterior IM cell types, and it is from these
populations that kidney organoids were generated.
11.2.3 Generation of Kidney Organoids from Human
Pluripotent Stem Cells
As described above, the final permanent kidney in the mammal arises from the
reciprocal interactions between the UB that branches from the nephric duct, a deriv-
ative of the anterior IM, and the MM, which is derived from the posterior IM
(Fig. 11.2). In mouse, lineage tracing using Eya1 confirms that while the mesen-
chyme of the metanephroi and the caudal region of the mesonephroi arise from an
EYA1+ population, the UB itself arises from an earlier OSR1+ population (Xu et al.
2014 ). Despite this, after induction of posterior PS and then IM, we have observed
the simultaneous formation of an epithelial population displaying markers of neph-
ric duct/UB (PAX2, GATA3, ECAD1, DBL) within a mesenchyme displaying
expression of MM/CM (SIX2, WT1, PAX2, HOXD11) (Takasato et al. 2014 ).
While both of these cell types arose in monolayer culture in defined media, with
time the cells within the dish reorganised into 3D clusters in which an epithelium
was surrounded by mesenchyme spontaneously forming CDH6+ early nephrons
(Takasato et al. 2014 ).
During normal kidney development, morphogenesis is a highly dynamic process
with cells migrating with respect to each other and in response to growth factor and
cell-cell-based signals (Short et al. 2014 ; Combes et al. 2016 ). This process occurs
in most tissues during development and is referred to as self-organisation. Individual
mesenchymal cells within each CM/ureteric tip swarm around the tip in response to
a variety of signals from the tip (Combes et al. 2016 ). In this way, the induction of
branching is maintained and the CM can reorganise around newly formed tips, con-
tinuing to reshape the growth factor field. This process undoubtedly also plays a role
in the positioning and induction of nephrons. Indeed, the process of cell-cell-
regulated self-organisation during vertebrate development is so strong that embry-
onic tissues can be dissociated down to single cells and reform structure after
reaggregation via self-organisation (Weiss and Taylor 1960 ). This is also the case
for the kidney (Unbekandt and Davies 2010 ; Lusis et al. 2010 ). As a result, we
reasoned that if the appropriate cell types were present in cultures of hPSCs
differentiated towards UB and MM, the provision of a 3D environment would
11 Recapitulating Development to Generate Kidney Organoid Cultures