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differentiation of human iPSC to a single renal cell type or segment (Song et al.
2012 ; Wang et al. 2013 ; Sharmin et al. 2016 ; Ciampi et al. 2016 ), but in this section
we will concentrate on the protocols (i) shown to establish multisegmented kidney
organoids and (ii) employing fully defined culture conditions (summarised in
Table 11.1).
Initial iPSC renal differentiation protocols were based on monolayer culture and
utilised various combinations of CHIR99021, BMP, FGF and activin signals.
Taguchi et al. ( 2014 ) applied a protocol developed for mouse ESC protocol to the
differentiation of human iPSC first generating embryoid bodies and subsequently
deriving appropriate MM (WT1+, PAX2+, SALL1+ and SIX2+) which, when cul-
tured with dorsal spinal cord, gave rise to contiguous WT+/nephrin + glomerular
structures, CDH6+ proximal tubules and CDH1+ distal tubules. The same group
later transplanted nephrin-GFP-expressing glomeruli underneath mouse renal cap-
sules and observed vascularisation of the iPSC-derived glomeruli by murine endo-
thelial cells (Sharmin et al. 2016 ). During their protocol optimisation, Lam et al.’s
(2014b) prolonged CHIR99021 exposure led to lateral plate mesoderm fate
(FOXF1+), but introduction of FGF2 signalling diverted CHIR99021-treated cells to
PAX2+ IM fate. They also demonstrated that FGF9 and activin treatment of IM gave
rise to SIX2+ MM and treatment of this population with further CHIR99021 induced
a downregulation of SIX2+ mesenchyme and the genesis of LTL+ proximal tubular
structures (Lam et al. 2014b).
Takasato et al. ( 2014 ) demonstrated both CHIR99021 and high BMP4/low
activin A could effectively induce PS mesendoderm but specified IM fate using
FGF9 and heparin which was continued to derive SIX2+ MM. Twelve days follow-
ing withdrawal of growth factors in CHIR99021-treated cell lines, both MM and UE
structures were present and self-organised when aggregated to form 3D structures
resembling kidney tubules expressing WT1, PAX2, CDH6, JAG1, aquaporins 1 and
2 and SLC3A1 (Takasato et al. 2014 ). While this first aggregate protocol utilised
foetal calf serum in its culture media, this group would later publish the refined
protocol described above to derive organoids displaying NPHS1+/WT+ glomeruli,
LT L+/CUBN+ PT, PAX2+/CDH1+/GATA3+ ureteric epithelium, CD31+ endothelium
and MEIS1+ stromal components (Takasato et al. 2015 ).
Self-organisation within the nephrogenic zone of the developing kidney is driven
by GDNF stimulation of UE branching morphogenesis by the MM and reciprocal
FGF9 and low-level Wnt signalling from the UB required for self-renewal and
maintenance of the cap mesenchyme. This self-organisation was also utilised by
Morizane et al. ( 2015 ) who published a protocol using 4-day CHIR99021+ noggin
to establish PS, followed by 3-day activin A to establish posterior IM and 7 day-
FGF9 to establish a SIX2+/WT1+/SALL1+ MM. Here nephrogenesis was also
induced by 2-day CHIR99021, giving rise to NPHS1+/PODXL+ podocytes, CDH2+/
LT L+ proximal tubules, CDH1+/UMOD+ loops of Henle and CDH1+ BRN1+ distal
tubule after 4-week culture. Where Takasato et al. ( 2015 ) pelleted differentiated
cells and cultured the pellets on an air-liquid interface, Morizane et al. ( 2015 )
replated cells in an ultra-low attachment, round bottom, 96-well plate. Freedman
et al. ( 2015 ) utilised a Matrigel sandwich culture technique to derive epiblast
M.H. Little et al.