Table 2.

Directed Differentiation of Human PSCs Into Kidney Cells

Study	Starting Substrate	Culture Method	Differentiation Protocol	Differentiation End Point	Findings
Schuldiner et al,⁵⁵ 2000	hESCs	EB formation	20% KOSR without LIF and bFGF (5 d) 20% KOSR + HGF or nerve growth factor or activin (10 d)		Expression of WT1 and RENIN by RT-PCR
Batchelder et al,⁵⁶ 2009	hESCs	EB formation Monolayer	10% FBS + RA/activin/ BMP7 (EB, 2 d) 10% FBS + RA/activin/ BMP7 (monolayer, 6 d)	PAX2⁺vimentin⁺ cells	Expression of PAX2, WT1, OSR1, EYA1, LHX1, CD24 by RT-PCR Expression of PAX2 and vimentin by IF
Lin et al,³⁴ 2010	hESCs	Monolayer	20% FCS (2 d) 5% FCS (12 d) Flow sorting by CD24⁺podocalyxin⁺ GCTM2⁻ cells	Intermediate mesoderm	Expression of WT1 and PAX2 by IF Up-regulation of renal developmental genes in microarray analysis
Song et al,⁵⁷ 2012	hiPSCs	EB formation Monolayer	2.5% FBS + activin/ BMP7/RA (EB, 3 d) 2.5% FBS + activin/ BMP7/RA (monolayer, 7 d)	Podocytes	Expression of podocin, synaptopodin, and PAX2 by IF Integration into WT1⁺ glomerular structures of dissociated- reaggregated E13.5 kidneys
Narayanan et al,⁵⁹ 2013	hESCs	Monolayer	REGM + 0.5% FBS + BMP2 + BMP7 Flow sorting for AQP1⁺ cells	Proximal tubular-like cells	Expression of AQP1 by IF and FACS Integration of AQP1⁺ cells into tubular compartments of ex vivo newborn mouse kidneys Formation of cord-like structures in Matrigel Parathyroid hormone–stimulated cyclic adenosine monophosphate production, GGT activity, ammonia production
Mae et al,²⁸ 2013	OSR1-GFP hiPSCs	EB formation Monolayer	Serum-free media + CHIR99021/activin (2 days) Serum-free media + CHIR99021/BMP7 (9–16 d)	Intermediate mesoderm	Expression of OSR1-GFP by FACS Expression of PAX2 and WT1 by IF Expression of kidney, adrenal gland, and gonadal markers by IF and RT-PCR Formation of growths with expression of kidney markers when injected into immunocompromised mice Integration into dissociated- reaggregated E11.5 mouse kidneys
Xia et al,²⁹ 2013	hESCs hiPSCs	Monolayer	Serum-free media + BMP4/FGF2 (2 d) Serum-free media + RA/ activin/BMP2 (2 d)	Intermediate mesoderm	Expression of PAX2, LHX1, OSR1, and WT1 by IF and RT-PCR Integration into ureteric bud structures of dissociated- reaggregated E11.5 kidneys
Taguchi et al,³¹ 2013	mESCs hiPSCs	EB formation	Serum-free media + activin (1d) Serum-free media + BMP4/CHIR (2.5 d) Serum-free media + activin/BMP4/CHIR/ RA/Y27632 (1 d) Serum-free media + CHIR/FGF9/Y27632 (2 d)	Metanephric mesenchyme	Nephron precursors derived from OSR1⁺ posterior IM and T⁺ posterior mesoderm Expression of Osr1, WT1, Pax2, Six2, and Sall1 by IF and RT- PCR Co-culture of induced EBs resulted in formation of glomerular-like and tubular-like structures expressing kidney markers Protocol reproducible in hiPSCs with longer time course of differentiation
Takasato et al,³⁰ 2013	hESCs	Monolayer	Serum-free media + activin/BMP4 or CHIR99021 (2 d) Serum-free media + FGF9 + heparin (4 d) Serum-free media + FGF9 + heparin (6 d) Serum-free media alone (6 d)	Metanephric mesenchyme	Expression of PAX2 and LHX1 by IF and RT- PCR (IM) Expression of SIX2, HOXD11, and WT1 by IF (MM) Formation of tubular-like structures expressing ECAD, JAG1, and CADH6 Integration into E12.5–E13.5 kidneys Self-aggregation into tubular-like structures expressing kidney markers
Lam et al,²⁷ 2013	hESCs hiPSCs	Monolayer	Serum-free media + CHIR99021 (1.5 d) Serum-free media + FGF2/RA (1.5 d) Serum-free media alone (6 d for tubular structures) or serum-free media + FGF9/activin (3–6 d for MM)	Intermediate mesoderm	Expression of PAX2, LHX1, and WT1 by IF Formation of polarized, apically ciliated tubular-like structures expressing LTL, KSP, and NCAD Tubular cells integrate into laminin-bounded structures of E12.5 kidneys Expression of SIX2, SALL1, and WT1 by IF with FGF9 treatment MM cells form LTL⁺ laminin⁺ structures in E12.5kidneys
Araoka et al,⁶³ 2014	OSR1-GFP hiPSCs	Monolayer	Serum-free media + CHIR99021 + AM580 or TTNPB (2 d) Serum-free media + AM580 or TTNPB (12 d)	Intermediate mesoderm	Expression of OSR1-GFP by FACS Expression of PAX2,WT1, LHX1, and SALL1 by IF Expression of kidney, adrenal gland, and gonadal markers by IF and RT-PCR Integration into dissociated- reaggregated E11.5 kidneys Formation of tubular-like structures in cell aggregates

FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; KSP, kidney-specific protein; LIF, leukemia-inhibitory factor; RT-PCR, reverse-transcription polymerase chain reaction.