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spheroids from iPSC and then induced epithelial-to-mesenchymal transition using a
short period of high-dose CHIR treatment (1.5 days, 12 μM) followed by culture in
B27- supplemented media. This resulted in the development of nephron structures
with WT1+/SYNPO+ podocytes, LTL+ proximal tubule, ECAD+ distal tubule and
CD31+ vascular progenitors which were subsequently used to model nephrotoxic
kidney injury and model disease (Freedman et al. 2015 ).
Imberti et al. ( 2015 ) omitted CHIR99021 from their protocol, instead using small
molecules CCG1423 (a Rho pathway inhibitor) and LY294002 (a phosphoinositol
3 kinase inhibitor). Activin A was added for days 2 and 3, guiding differentiation to
a T+/OSR1+ primitive streak, and another 6 days of small-molecule exposure
achieved a WT1+/PAX2+ IM. The stage 2 protocol for the differentiation of a WT1+/
PAX2+/SIX2+/SALL1+ MM/nephron progenitor population consisted of 6-day cul-
ture in FGF2, BMP7 and GDNF. The role of GDNF in this culture is unclear given
the absence of collecting duct, and the work in mouse would suggest that the addi-
tion of BMP7 may induce differentiation rather than maintaining MM/CM (Brown
et al. 2013 ). Indeed, across the time course investigated in this study, expression of
SIX2 was maximal at day 6 but subsequently declined. The outcome of the differ-
entiation was characterised as glomerular (CD133+/CD24+/claudin1+) and proximal
tubule (AQP1+/GGT1+) (Imberti et al. 2015 ). Araoka et al. ( 2014 ) also used small
molecules AM580 and TTNPB identified during high-throughput screening using
an OSR1-GFP reporter hiPSC line, but in contrast Imberti et al. ( 2015 ) utilised
CHIR99021 instead of activin A to induce IM and demonstrated OSR1+/WT1+/
PAX2+/SALL1+ IM monolayer which formed LTL+/AQP1+ proximal tubular cells,
PDX+/WT1+ podocytes and DBA+/CK8+/SALL4+ nephric duct alongside adrenal
cortex and gonad markers.
In summary, despite the variety of published protocols, the consensus approach
from the field currently favours the induction of Wnt and/or activin signalling to
derive PS and subsequently drive nephrogenesis followed by FGF signalling to pro-
mote maintenance of a MM cell mass. The ability of cells to self-organise in mono-
layer and 3D culture suggests that once a certain stage of differentiation is reached,
cells can guide their own differentiation and maintenance, as well as that of the
cultured tissue. Future challenges in protocol development will include the matura-
tion and vascularisation of glomerular structures as well as improving the maturity
of tubular expression and collecting duct derivation.
11.4 Applications to the Use of Kidney Organoids
11.4.1 Utility of Kidney Organoids for Analysing Human
Kidney Development
While there has been some limited anatomical studies of the developing human kidney
(Osathanondh and Potter 1963 ), and more recently some analysis of gene expres-
sion using in situ hybridisation and immunohistochemistry (O’Brien et al. 2016 ),
M.H. Little et al.