Table 1.

The summary of the protocols for the differentiation of stem cells into the nephron progenitor (NPs) and the podocyte progenitors

Original cells	W9.5 ESCs (P18) derived Pax2-GFP+ mESCs	mESCs derived EBs	hiPSCs derived from human kidney mesangial cells	H6 cells (CD-1 mouse’s Pax2+ KSCs)	HK2 (Human kidney cell line-adult proximal tubule cells)
Number of cells	EBs from W9.5 ES cells (P18) (1 × 105/ml, 2 × 103/ml), EBs derived Pax2-GFP+ ESCs (8 × 104/ml)	-	-	-	-
Differentiation culture conditions (small molecules/growth factors/transcription factors/micro RNA)	Stepwise: ME: DMEM, 1% methylcellulose, BMP4 (2 ng/ml) IM: BMP2, BMP4, BMP7 each with (0.5, 5, 50 ng/ml), Chordin (1.25 µg/ml), BMP2, BMP4, BMP7 (5 ng/ml), serum & serum free conditions	Stepwise: mESCs on mouse feeder layer then on 1% agar within 2 days to form EBs then for 6 days trypsinized EBs on 0.1% gelatin, DMEM, 15% FCS, L-glutamine (2 mM), β-Mercaptoethanol (5 × 10-5 M), NEAA (1:100), RA (10-6, 10-7, 10-8), A A (1, 5, 10 ng/ml), 10th day, UB cells of gestation day 13 fetal kidney conditioned medium for further 10 days	DMEM-F12, 2.5% FBS, β-Mercaptoethanol (100 µM), 0.1% gelatin coated dishes. AA (10 ng/ml), RA (0.1 µM), BMP7 (15 ng/ml)	High glucose DMEM, 2 mM glucose, 20% FBS, No other molecules. Spontaneous differentiation	GFP-Lentivirus for 7 days + VPA (2 mM). Combinatorial screening of 15 different factors for NPs (Six1, Six2, Pax2, OSR1, CRYM, N-NYC, cMYC, HOXA11, EYA1, SNAI1, SNAI2, WT1, MEOX2, HMGA2, OCT4)
Endpoint duration of analysis	16th & 19th day	10th & 20th day	•SEM on 10th day	4 weeks	6 days
			•Expension of differentiated cells + 10 days without small molecules & growth factors
Detection methods/characterization	•ICC (Pax2+ in EBs)	•ICC IM: (Brachyury, Pax2) Renal genes: (WT1, E-CDH, POD-1, DB, Pax2)	•SEM	•ICC (Pax2, WT1, αSMA, CK8, GDNF, Musahi1, Nanog)	•ICC (10 pools were identified based on induction of CITED1. further screening identified pool 8 by qRT-PCR)
	•qRT-PCR (α-Globin, β-h1globin, Aqp1, Brachyury, GDNF, GFP, HPRT, CDH16, LacZ, Mineralocorticoid receptor, NPHS1, OCT4, Pax2, PODXL, Pod-1, Wnt4, Wnt11, WT1)	•Flow cytometry (Brachyury, Pax2)	•ICC (Nephrin, Podocin, Pax2, WT1, SYNPO)	•RT-PCR (Pax2, WT1, GDNF, Sall1, PODXL, SYNPO, UMOD, Desmin, Megalin, AQP1, AQP2, CK8, Six2, Nanog)	•qRT-PCR (Six1, Six2, OSR1, Pax2, HOXA11, EYA1, SNAI2, CITED1, E-CDH, MMP2, MMP9)
	•FACS (Pax2 GFP+, PI)				•RT-PCR (Six2, CITED1, Sall1, FOXD1, GSC, FOXA2, PAX6, NANOG)
					•ICC (Pax2, CITED1)
Functional analysis	-	-	•Cell contractility assay (AII, RFP-actin, RFP-talin)	•Alkaline phosphatase staining for proximal tubular cells	•Recombination assay E12.5 mouse kidney + GFP+ single cells, detected by
			•Cell permeability assay {FIT-C labeled albumin (0.5 mg/ml)}	•No specific functional assay for podocytes like cells	•ICC (WT1+, Six2+, Calbindin-)
			•Re-aggregation assay (mouse embryonic kidneys, E 13.5-15.5)	•Flow cytometry for stem cells markers (Sca1+, CD24+), mesenchymal markers (CD29, CD44) hematopoietic markers (CD45, CD117)
Uni/multi progenitors	Multipotent progenitor from IM to renal lineage cells	Multipotent progenitor from IM to renal lineage cells	Unipotent, podocyte progenitors	Multipotent progenitor to podocytes, mesangial cells, & proximal tubular cells.	Nephron progenitors
References	[7]	[8]	[11]	[21]	[22]
Original cells	hESCs {HES3 (MIXL GFP/wt)}	hESCs-H9 cell line, hiPSCs-derived from CRL2097 fibroblast	hPSCs {iPSCs (Fibro-epi)}, ESC (H1) human fibroblast episomal derived	hiPSCs (SC101A-1) clone IV derived EBs	Sprague Dawly rat’s BM-MSCs, AD-MSCs
Number of cells	12000-15000 cells/cm2	-	~300-500 cells/colonies	-	-
Differentiation culture conditions (small molecules/growth factors/transcription factors)/micro RNA)	Stepwise: PS: 2-3 days matrigel coated dishes, 1. BMP4 (30 ng/ml), AA (10 ng/ml), or 2. BMP4 (30 ng/ml), CHIR99021 (8 µM), serum free APEL media. IM: 4 days in FGF9 (200 ng/ml), heparin (1 µg/ml). further differentiation 4-11 (6 days) for 1. FGF9 (200 ng/ml), BMP7 (50 ng/ml), RA (0.1 µM), Heparin (1 µg/ml). 6 days For 2. FGF9 (200 ng/ml), Heparin (1 µg/ml). Cultured for further 6 days	Stepwise: Serum & feeder free system	Stepwise: IM: 2 days on growth factor reduced matrigel-coated plates, DMEM-F12, BSA (17.5 mg/ml), hInsulin (17.5 µg/ml), h holo-transferin (275 µg/ml), Monothioglycerol (450 µM), L-glutamine (2.25 mM), NEAA (2.25 mM), Penicillin (100 units/ml), Streptomycin (100 µg/ml), bFGF (50 ng/ml), hBMP4 (30 ng/ml), For further 2 days in ATRA (1 µM), hAA (10 ng/ml), BMP2 (100 ng/ml) + same medium	Stepwise: ME: 0-3 days, IM: 3-6 days, MM: 6-12 days, NPs: DMEM-F12, 5% FBS, NEAA (0.1 mM), β-Mercaptoethanol (0.1 mM), ATRA (0.1 µM), CCG1423 (1 µM), LY294002 (5 µM) till day 6; AA (10 ng/ml) for day 2-4. For 6-19 days, BMP7 (50 ng/ml), FGF2 (10 ng/ml), GDNF (15 ng/ml)	Fibronectin coated dishes, DMEM low glucose, 15% FBS, FGF2 (50 ng/ml), TGFβ2 (4 ng/ml), LIF (20 ng/ml)
		PS: 3 days {1 day, AA (100 ng/ml), Wnt3a (100 ng/ml), IM: 2 days, BMP4 (20 ng/ml), bFGF (10 ng/ml)} IM: 6-8 days, RA (10 µM), BMP7 (50 ng/ml), bFGF (10 ng/ml). 15 days NP: BMP7 (150 ng/ml), bFGF (50 ng/ml). For all above, the medium RPMI-1640 containing 2% B27, L-glutamine (2 mM), 1% PenStrep.
		For podocytes differentiation NPs cultured on fibronectin-coated dishes in VRAD medium (DMEM-F12, RA (100 µM), 10% FBS) for 7 days
Endpoint duration of analysis	2-18 days	15 days NPs. Later Podocyte formation	1-6 days	0, 6, 12, & 19 days	7 days
Detection methods/characterization	•FACS PS: {Post 3 days, (2 × 106 Cells) MIXL1-GFP+}	•qRT-PCR PS: (T, MIXL1, EOMES) Endodermal: (SOX17, FOXA2) Ectodermal: (PAX6, SOX1) IM: (OSR1) NPs: (SIX2, WT1, GDNF, HOXDA11) Metanephric stroma/UB: (FOXD1, HOXB7. Bone, RUNX2, COL1A1) Vascular endothelium: (PCAM1, TIE2) Smooth Muscle: (MYH11, CALPONIN) Liver: (ALB, AAT) Neuron: (TUJ1, MAP2) Tubular: (SLC12A3, CD13, AQP1) Podocyte: (SYNPO, Nephrin)	•qRT-PCR IM: {T, OSR1, LHX1(LIM1), Pax2, Pax8, GATA3, OCT4, Nanog, SOX3} IM ureteric progenitor-like cells: (Six2, GDNF, WT1, Sall1, Cited1) UB: (HOXB7, RET, GFRA1)	•ICC (OSR1, WT1, Pax8, Pax2, Six2, Sall1, CD133, CD24, NCAM, Claudin1, AQP1, GGT1, SSEA4, TRA1, Nanog, T, AFP, Pax6, Nkx2.5)	•ICC (Wnt4, WT1, Pax2, Vim, Oct4, Sox2, E-CDH, ZO-1)
	•ICC (Pax2, OSR1, LHX1, TBX6, SOX17, Six2, E-CDH, WT1, HOXD11, GATA3, JAG1) In this quantification proportion of induced cells (Pax2, LHX1, Sox17, Six2, WT1, CDH6, SYNPO)	•RT-PCR IM: (OSR1, PAX2, SALL1, EYA1, WT1) NPs: (SIX2, CITED1, OSR1, PAX2, SALL1, EYA1)	•ICC IM: (HuNu, CK8, Six2) IM ureteric progenitor-like cells: {(Six2, WT1, LHX1 (LIM1)}	•FACS (TRA-1-81)	•qRT-PCR (Pax2, Wnt4, WT1, E-CDH, ZO-1)
	•qRT-PCR, day 3 PS: {SOX17, Brachyury (T) MIXL1} day 6 IM: (Pax2, LHX1, FOXF1, TBX6)	•ICC (T, TRA1-81, OCT4, OSR1, PAX2, SALL1, SIX2, WT1, E-CDH, ZO1, KRT18, F-ACTIN, CD13, AQP1, MUCIN1, SYNPO, PODXL)		•qRT-PCR human (POU5F1, Nanog, DNMT3B, GABRB3, GDF3, SOX2, TDGF1, RAF1, ELF1, T, LHX1, OSR1, SIX2, PAX8, NANOG, SALL1, WT1, PAX2)
	•RT-PCR (Pax2, LHX1, OSR1)
	•RT-PCR for day 0-17 PS: (MIXL1, LHX1) IM: (LHX1, Pax2, OSR1) MM: (OSR1, SIX2, WT1, GDNF, HOXD11) UE: (PAX2, CRET, HOXB7) Ectoderm: (PAX6)
	•Pellet IMF (CALB1, AQP1, AQP2, SLC3A1, HuMt, HuNu)
Functional analysis	•3D culture (10 × 105 cells), collagen IV coated (10 µg/cm2) filter membrane (0.4 µm)	•Alkaline phosphatase staining for tubular cells	•3D organ co-culture assay	•Cisplatin-induced AKI model	-
	•Re-aggregation assay, embryonic kidneys (12.5-13.5 dpc) collagen IV coated (10 µg/cm2) filter membrane	•In vitro tubulogenesis assay	•qRTPCR for above {(T, OSR1, HOXB7, LHX1 (LIM1), Pax2, GFRA1)}	•HC (H&E, PAS)
		•No functional assay for podocytes	•ICC (CK8, HuNu, Six2, ZO1)	•IHC (HNA, hMitochondria, AQP1, WGA lectin, PNA lectin, AQP3, Ki67)
Uni/multi progenitors	Multipotent progenitors, ureteric & metanephric progenitors	Multipotent progenitor to podocytes, & tubular cells	Ureteric bud committed renal progenitor-like cells	Multipotent progenitor	Nephron progenitors
References	[9]	[10]	[12]	[14]	[19]