TABLE 1.
Major approaches and protocols for directed differentiation of hPSCs into renal progenitor cells and kidney organoids.
| Author and year | Stem cell line | Stepwise differentiation protocol | hPSC-derived renal cells by 2D or EBs differentiation | Generation or kidney organoids | Cell types within kidney organoids | Functional and validation assays | ||
|---|---|---|---|---|---|---|---|---|
| Song, B 2012 | hiPSCs from human kidney mesangial cells | 3days suspension culture of iPSCs colonies with DMEM-F12 + 2.5% FBS + AA + BMP7+RA | Podocyte progenitor cells expressing Podocin, Synaptodin, PAX2, WT1, NPHS1 | N/A | N/A | Permeability assay showed endocytic uptake of FITC- albumin. | ||
| 7–8 days on gelatin coated monolayer with same suplementation | Integration within glomerular aggregates of embryonic mouse kidney explants. | |||||||
| Narayanan, K 2013 | (HUES)-7 hESCs | 20 days culture on Matrigel with REGM +0.5% FBS + BMP2+BMP7 | Renal proximal tubular-like cells expressing AQP1 and a markers profile similar to HPTCs | N/A | N/A | Contribution to CK18-positive tubular epithelia when injected into mouse cortex kidney explants. | ||
| Formation of tubular structures after implantation subcutaneously into immunodeficient mice. | ||||||||
| Response to PTH and GGT activity into biorreactor cultures for further bioartificial kidney applications. | ||||||||
| Mae, S 2013 | 201B7 hiPSCs from dermal fibroblasts | Stage 1: 2days AA + CHIR | DMEM/F12 + 2%FBS DMEM/F12 + B27 | EB method Colony method (Matrigel) Single-cell method (Collagen I) Serum-free single-cell method (Collagen I) | IM cells expressing OSR1 that further differentiated into polarized tubule-like structures expressing LTL/LAMININ/AQP1, few UB cells expressing DBA, glomerular cells expressing PODXL/PNA and gonadal or adrenocortical cells expressing GATA4/GATA6/HSD3β | N/A | N/A | Differentiation of IM cells into renal tubular like cells after their transplantation into the epididymal fat pads of immunodeficient mice |
| Stage 2: 8days BMP7+CHIR | DMEM/F12 + 10%KSR DMEM/F12 + B27 | EB method Colony method (Matrigel) Single-cell method (Collagen I) Serum-free single-cell method (Collagen I) | Integration to form tubular like structures when cocultured with mouse metanephric tissue. | |||||
| Xia, Y 2013 | H1 hESCs and 201B7 hiPSCS from dermal fibroblasts | Monolayer cell culture on matrigel and DMEM/F12 supplemented with: 2days BMP4+FGF2 and insulin → Mesoderm | UB progenitor-like cells expressing PAX2/LHX1 and a markers profile similar to UB | N/A | N/A | Integration into the UB tip and truck when re-associated with mouse embryonic kidneys | ||
| 2days RA + AA and BMP2 → IM and UB lineages | ||||||||
| Takasato, M 2014 | HES3 hESCs, H9 hESC and CRL2429 C11 hiPSCs from foreskin fibroblasts | Matrigel culture and APEL basal media suplemented with: 2days AA BMP4 → PS 4days FGF9 → IM 11dayays FGF9+BMP7 and RA → MM and UE | Stepwise differentiation from PS cells expressing MIXL1/LHX1 to IM cells expressing OSR1/PAX2/LHX1 to finally obtain both: MM cells expressing SIX2/WT1/GDNF/HOXD11 and UE cells expressing C-RET/HOXB7 | N/A | N/A | Reaggregation assays with dissociated mouse embryonic kidneys showed specific integration into only MM and UE compartments | ||
| Later differentiation timepoints showed UE structures expressing ECAD surrounded by MM cells | ||||||||
| Matrigel culture and APEL basal media suplemented with: 2days CHIR → PS 10days FGF9 → IM 6days No growth factors → MM and UE | Same stepwise differentiation but with a faster induction of kidney markers and a more prolonged expression of MM genes. | Enzymatically dissociation into single cells, pelleted and cultured on a filter membrane at an air–media interface with DMEM 10% FCS during 4days. | ECAD + tubules expressing UE markers PAX2/AQP2 and proximal tubule markers AQP1/SLC3A1. | Reaggregation assays with dissociated mouse embryonic kidneys showed integration into compartments of the developing kidney including UE, early nephron/renal vesicles and nephron progenitor mesenchyme | ||||
| Additional GATA3 expression in UE cells already expressing PAX2 | ||||||||
| MM cells expressing PAX2 and condensing tightly around the UE tips. Presence of renal stroma cells WT1- and HOXD11+. | MM cells expressing WT1/PAX2 and surrounding the ECAD + UE. MM also contained renal vesicles expressing JAG1/ECAD. | |||||||
| Later differentiation showed UE structures expressing ECAD and surrounded by MM. This MM contained nephron/renal vesicles like structures expressing CDH6/JAG1+. | ||||||||
| Lam, A. Q. 2014 | H1, H9, CHB8-H2B-GFP hESCs and hiPSCs from human foreskin or human dermal fibroblast | Geltrex culture and RPMI basal media suplemented with: 2days CHIR → ME 4days FGF2+RA → IM 5days FGF9+AA or 7days No growth factors | Stepwise differentiation from mesendoderm cells expressing T/MIXL to IM cells expressing PAX2/LHX1 that: | After growth factors withdraval spontanously induced tubule-like structures expressing LTL/KSP/NCAD | N/A | N/A | Re-aggregation assays of tubular-like cells with dissociated mouse embryonic kidneys showed integration in both metanephric interstitium and murine laminin-bounded structures but not in murine tubular-like structures. | |
| In vivo implantation of tubular cells beneath the kidney capsule of immunodeficient mice generated human growths expressing AQP1. | ||||||||
| After FGF9 + AA treatment induced cap mesenchyme NPCs expressing SIX2/SALL1/WT1 | CHIR treatment in SIX2+ cap mesenchyme nephron progenitors induced tubulogenesis similar to an in vivo scenario. | |||||||
| Re-aggregation assays of SIX2+ cap mesenchyme NPCs showed organization into clusters of cells expressing LTL | ||||||||
| Taguchi, A 2014 | Based in protocol for mouse ESCs, hiPSCs from dermal fibroblasts were differentiated | Suspension culture and: 1day Serum free medium with BMP4+Y27632 → EBs 2days Serum free mediumwith AA + Fgf2 → Epiblast 6days BC10 medium with BMP4+CHIR → Posterior Nascent M 2days ABC3R medium with AA + BMP4+CHIR + RA→ PIM 3days ABC3R media with CHIR + FGF9 → MM | Majority of IM cells expressing WT1 at day 11. | Reagregation on day 14 with mouse embryonic spinal cords and cultured on a air-fluid interface of a polycarbonate filter | Early glomeruli expressing WT1/NPHS1. | Chimeric orgnaoid was used as a functional read out | ||
| Proximal tubules expressing CDH6. | ||||||||
| MM cells expressing WT1/PAX2/SALL1 at day 14. | Pistal tubules expressing CDH1 structures. | |||||||
| Some MM cells expressing SALL1/PAX2. | ||||||||
| Imberti, B 2015 | SC101 A1 hiPSCs from foreskin fibroblasts and in house derived hiPSCs from human fibroblast | DMEM/F12 supplemented with: 1day RA + PI3K inhibitor + RhoA inhibitor 2days RA + PI3K inhibitor + RhoA inhibitor + AA → ME 3days RA + PI3K inhibitor + RhoA inhibitor → IM 13days FGF2+BMP7+GDNF → MM differentiating into NPCs | MM cells expressing WT1/PAX8/PAX2/SIX2/SALL1 that progressively adquired renal progenitor phenotype specific markers such as NCAM, CD133, CD24 or AQP1 and formed nephrogenic like peripheral patterns. | N/A | N/A | Day 12-differentiated cells were intravenously infused into cisplatin-induced AKI mouse model. Four days after infusion, human cells were found integrated into proximal murine tubuli and a significant improvement of renal function in terms of reduced BUN levels and improved histological observations such as reduction of cell swelling, less cast deposition, integrity of brush borders and reduced cell necrosis | ||
| Morizane, R 2015 | H9 hESCs, hiPSCs from dermal fibroblasts | Culture on Geltrex and Advanced RPMI 1640 suplemented with: 4days: 8 CHIR for ESCs; CHIR + noggin for iPSCs. → LPS 3days: AA → PIM 2days: FGF9. Next steps on 2D or in 3D kidney organoids → MM 2days: CHIR + FGF9 → Pretubular aggregate 3days: FGF9 → RV 7–14days: no growth factors → Nephrogenesis | NPCs expressing SIX2/SALL1/WT1/PAX2 on day 9 of differentiation that further differentiated on day 11 in RVs expressing PAX8/LHX1/HFN1b/BRN1. RVs finally developed into elongated epithelial nephron structures including glomerular podocytes expressing NPHS1/PODXL, proximal tubules expressing LTL/CDH2 and loops of Henle/distal tubules expressing ECAD/CDH1/UMOD/BRN1 | Alternatively to 2D differentiation on day 9, NPCs were replated in ultra-low attachment round bottom plates, pelleted and mantained in 3D suspension culture. Spherical aggregates underwent nephrogenesis. | Clusters of podocyte-like cells expressing WT1/PODXL/NPHS1 connected to tubular structures with proximal tubules expressing LTL/CDH2/AQP1, loops of Henle expressing CDH1/UMOD and distal tubules expressing only CDH1. | Supression of proximal tubules by Notch signalling inhibitor DAPT as previous developmental studies in mice. | ||
| Tubular toxicity response to cisplatin and gentamicin. | ||||||||
| Freedman, BS (2015) | H9 and WA09 hESCs, hiPSCs derived from foreskin and dermal fibroblasts. | Sandwiched culture between matrigel 3days: mTeSR1 medium → Cavitated spheroids 1.5days: RPMI suplemented with CHIR → ME 11.5days: RPMI suplemented with B27 → from MM to RVs Onwards no growth factors → Mature tubular organoids | Full protocol carried on 3D culture: from cavitated spheroids to kidney organoids. | hPSCs were dissociated and sandwiched between two layers of dilute Matrigel. Embebed colonies were induced to form cavitated spheroids that further differentiated into kidney organoids. | Sequential presence of IM cells expressing PAX2 on d7, MM expressing SIX2/WT1 on d14 and renal vesicles expressing PAX2/LHX1 on d22 that final differentiated into renal cell types including podocyte-like cells expressing WT1/PODXL/SYNPO, proximal tubular cells expressing CDH1/LTL, distal tubular cells tubules expressing only CDH1 and endothelial cells expressing CD31/vWF+. Off-target neuron-like cells expressing TUJ1 were also observed. Tubules expressed nephron progenitor/renal vesicle markers including LHX1 and PAX2. MM cells expressing SIX2 appear adjacent to organoids. | Selective uptake of dextran cargoes in tubules. | ||
| Tubular toxicity response to cisplatin and gentamicin. Disease modelling when PKD1, PKD2 and PODXL are depleted by CRISPR. | ||||||||
| Implantation of organoid cells into the cortex of immunodeficient mice showed 3 weeks survival and expression of LTL at similar intensities as neighboring mouse tubules. | ||||||||
| Takasato, M 2015 | CRL1502-C32 hiPSCs derived from fibroblasts | Culture on matrigel and APEL basal medium suplemented with: 4days: CHIR → Anterior and posterior IM 3days: FGF9+heparin → IM 11–18days: CHIR for 1 h, then 5days FGF9+heparin and finally no growth factors → Nephrogenesis into self-organizing organoid. | Posterior IM cells expressing GATA3/HOXD11 on day 7. Alternatively to organoid generation cells were further differentiated in 2D to UE expressing GATA3/PAX2/ECAD or MM expressing only PAX2 and its derivatives expressing PAX2/ECAD. Preferential induction of UE vs. MM was tested with different extents of CHIR treatment in combination with different growth factors. | On day 7 cells were dissotiated, pelleted and transferred to a pore polyester membrane of a transwell culture system. | Early podocytes expressing WT1/NPHS1. Proximal tubules expressing CDH1/LTL. Early loops of Henle expressing CDH1/UMOD. Distal tubules expressing only CDH1 that were associated with a CD network expressing PAX2/GATA3/ECAD. Cortical stroma cells expressing FOXD1/MEIS1. Medullary stroma cells only expressing FOXD1. Endothelial cells expressing CD31/KDR/SOX17. | Selective uptake of dextran cargoes in tubules. | ||
| Tubular toxicity response to cisplatin. | ||||||||
| Comparative RNA-Seq analysis clustered kidney organoids at d11 and d18 with first trimester human fetal kidney. | ||||||||
| TEM showed the presence podocyte-like cells aligned on a basement membrane and developing primary and secondary cell processes. | ||||||||
| Taguchi, A 2017 | 201B7 hiPSCs from dermal fibroblast | NPCs induction from hiPSCs adapted from Taguchi, et al., 2014 1day Aggregation in V-bottom plates Y27632 + AA + bFGF → EBs 6days in U-bottom plates CHIR+Y27632 → M 2days ABC3R medium with AA + BMP4+CHIR + RA+Y27632 → PIM 3days CIF medium with CHIR + FGF9+Y27632 → MM | NPCs defined by positive expression of ITGA8 and negative for PDGFRA expression. | N/A | N/A | Reconstitution assays of induced NPCs and UB cells from mESCs with primary stromal progenitors generated murine organoids with nephrons interconnected by branched epithelium. These assays are NOT characterized with human cells in this report. | ||
| UB induction and WD maturation from hiPSCs in basal media DMEM/F12/B27 supplemented with: 1day Y27632 + AA + BMP4 in V-bottom plates → EBs 1.5days CHIR+BMP4 in U-bottom plates → M 2days RA + FGF9+LDN193189 + SB431542 → Anterior IM 2days RA + CHIR + FGF9+LDN193189 → WD 2days Y27632 + RA + CHIR + FGF9+FGF1+LDN193189 + Matrigel → 3D WD maturation 2days previous components + GDNF → 3D WD maturation 2days previous components without FGF9 → UB organoid |
WD precursors expressing CXCR4 and KIT markers | Sorting of CXCR4+/KIT+ and aggregation in V-bottom plates at day 6.5 of differentiation. For branching, UB organoids on day 12.5 were embedded in transwell inserts and cultured in DMEM/F12 suplemented with Matrigel+10%FBS + RA + humanRspondin1+GDNF + FGF1+FGF7+LDN193189 | UB cells expressing HNF1b, E-cadherin, and CALB1 that generated a branched organoid. Ureteric epithelium showed tips with cells expressing SOX9 and stalk regions with cells expressing CK8. Detailed analysis of the tip region identified the typical ampulla or dichotomous bifurcation with cells expressing PAX2/ECAD. | Evaluation of PAX2 knockout hiPSCs for NPCs or UB induction elucidated the role of this transcription factor into MET of WD precursors. | ||||
| Garreta, E 2019 | ES[4], H1, H9 hESCs and CBiPSsv-4F-40 hiPSCs derived from CD133+ cord blood cells | Initiation in VTN coated plates. Advanced RPMI 1640 basal medium was suplemented with: 4days CHIR → PPS 1day AA + FGF9 → IM Generation of IM commited spheroid 3days FGF9+CHIR →→ NPCs 5days FGF9 → RV 8days No growth factors → Nephrogenesis | PPS cells expressing T that further after CHIR treatment that further differentiated into IM commited cells expressing PAX2 and other markers such as OSR1/HOXD11/GATA3 | IM committed cells were dissociated and agregated into low attachment round bottom plates for organotypic culture | Just after final FGF9 treatment organoids presented patterned RVs with cells expressing PAX2/WT1/LHX1/PAX8/HNF1β/ECAD/BRN1. | TEM showed the presence podocyte-like cells with deposition of a basement membrane and developing primary and secondary cell processes. Presence of brush borders and high mitochondrial content was observed in epithelial tubular-like cells. | ||
| Comparative RNA-Seq analysis clustered kidney organoids with second trimester human fetal kidney. | ||||||||
| RVs finally developed at the end of differentiation into nephron-like structures that were segmented into proximal tubules expressing LTL/AQP1/SLC3A1 loops of Henle expressing ECAD/UMOD distal tubules only expressing ECAD+ glomeruli expressing PODXL/PODOCIN/NPHS1/WT1 | Reagregation assays with mouse embryonic kidney cells showed how differentiated cells after CHIR + FGF9 treatment integrated into mouse nascent nephron structures. | |||||||
| Recapitulate complex nephron patterning events such as distalization after b-catenin induction or severe loss of glomeruli and proximal tubuli after Notch signalling disruption. | ||||||||
| Enhanced tubular differentiation under organoid culture favouring oxidative phosphorylation. | ||||||||
| Implantation of organoids into CAM promoted vascularization which enhanced maturation of nephron structures and was able to deliver injected chemicals such as cisplatin. | ||||||||
| Organoid differentiation was enhanced when IM commited cells were differentiated in softer substrates similar to the stiffness of in vivo microenviroments. | ||||||||
| Low, J. H. 2019 | H9 and H1 hESCs and GM10287 hiPSCs derived from fibroblasts | Culture on Matrigel and Advanced RPMI 1640 medium suplemented with: 4days CHIR → PS 3days without growth factors → IM 2days FGF9+CHIR → NPCs 1day FGF9+Y27632 → Nephrogenic organoids 9days FGF9 → Vascularized Kidney organoids | PS cells expressing T/MIXL1 that differentiated to IM cells expressing HOXD11/WT1 which finally generated NPCs expressing SIX2/SALL1 | NPCs on day 10–12 were dissociated, aggregatedand pelleted in U-bottom plates. | Transient pretubular aggregates expressing SIX2/SALL1/LHX1/PAX8 that further developed into nephron containing: podocytes expressing NPHS1/PODXL/VEGFA, proximal tubular cells expressing LTL, medial tubular cells expressing JAG1 and distal tubular cells expressing CDH1. | Coculture with HUVECs showed integration of exogenous endothelial cells with resident vascular network increasing organoid vascularization. | ||
| VEGFR inhibitors compris∫ed organoid vascular networks | ||||||||
| Vascular progenitors expressing KDR that further adquired CD31/CD34 expression and placed alongside nephron structures. | scRNA-seq suggested a subset of NPCs with vascular progenitor-like property as the origin of organoid vasculature. | |||||||
| CHIR treatments altered neprhon patterning by promoting tubulogenesis. | ||||||||
| In vitro dextran uptake by proximal tubules. | ||||||||
| Implantation beneath the renal capsule of immunocompromised mice improved organoid differentiation and vascularization. In vivo filtration and reabsorption by implanted organoids was shown after perfusion of dextran into murine vasculature. | ||||||||
| Cystogenesis recapitulation by kidney organoids differentiated from ARPKD hiPSCs | ||||||||
| Yoshimura, Y 2019 | 201B7 hiPSCs derived from fibroblasts and RN7 derived from blood cels | Starting from NPCs differentiated from hiPSCs as Taguchi, A 2017 protocol. NPCs ITGA8+/PDGFRa- population was sorted and cultured in U-bottom plates with serum free medium suplemented with: 1day CHIR + Y27632 → Pretubular aggregate aggregated cells were transferred to a transwell insert 2days FGF9+IWR-1+SB431542 + RA → Proximalized RV 6-9days IWR-1+SB → Podocytes | NPCs first differentiated in pretubular aggregates expressing LHX1 that further acquired CDH1 expression generating RVs. RVs differentiated up to 90% of podocytes expressing NPHPS1. | N/A | N/A | RNA-seq showed similar gene expression profile to human adult podocytes. | ||
| Induced podocytes also showed higher protein expression levels of podocyte-related proteins than inmortalized podocytes or podocytes derived from convectional kidney organoids. | ||||||||
| PAN treatment induced podocytes injury as in vivo. | ||||||||
| TEM showed protrusions of the basolateral domain expressing NPHS1 on their surface membrane. Slit diaphragm–like structures were also detected. | ||||||||
| Kumar Gupta, A 2020 | H9 hESCs and WTC11 hiPCs derived from fibroblasts | Starting from NPCs differentiated from hiPSCs as Morizane et al., 2015 protocol. Organoids were generated and cultured in air-liquid interface with APEL2 basal medium suplemented with: 4days BMP7+FGF9+Heparin 7days No growth factors | N/A | NPCs derived by Morizane protocol were dissociated, aggregated in a small volume which was finally dropleted on polycarbonate filter to generate a air-liquid interface culture system. After two days, differentiating organoids were mixed with newly differentiated NPCs and reaggregated to finally generate heterochronic organoids. Control organoids were not mixed with new NPCs | Proximal tubuli expressing LTL distal tubuli expressing BRN1/CDH1 collecting duct expressing CDH1/GATA3/DBA Podocytes expressing PODXL/WT1 endothelial network expressing CD31 Pericytes expressing PDGFRβ Remarkably heterocronic organoids displayed the double number of structures stained for each molecular marker and showed less remnant undifferentiated NPCs expressing SIX2 than control organoids. | Engrafted heterocronic organoids under the kidney capsules of immunocompromised mice showed enhanced maturation and functional vascularization. | ||
| Systemic perfused FITC-IB4 labeled endotelial vascular cells in close contact with organoid podocytes. | ||||||||
| Dextran was accumulated in organoid tubules after after its systemic perfusion. | ||||||||
Comparison of recent protocols that have been used to generate renal cells and eventually kidney organoids from hPSCs, including the following information: Schematic description of protocol steps in terms of duration, growth factors and lineages obtained, Renal cells induced by each protocol prior organoids formation in two-dimensional or Embryonic Body differentiation prior to organoids generation; Description of the method used to generate kidney organoids in vitro; Type of renal cells and structures appearing into organoids and finally; Assays performed to either improve the characterization or validate functionally the renal derivates obtained after each differentiation protocol. AA, activin A; AKI, acute kidney injury; AQP1, aquaporin 1; AQP2, aquaporin 1; ARPKD, autosomal recesive policystic kidney disease; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BRN1, POU, Class 3 Homeobox 3; BUN, blood urea nitrogen; CD, collecting duct; CD133, cluster of differentiation 133; CD24, cluster of differentiation 24; CD31, cluster of differentiation 31; CD34, cluster of differentiation 34; CDH1, cadherin 1; CDH2, cadherin 2; CDH6, cadherin 6; CHIR, CHIR99021 inhibitor; CK18, cytokeratin 18; CXCR4, C-X-C chemokine receptor type 4; d, days; DBA, lectin Dolichous biflorus agglutinin; EB, embryoid body; ECAD, E-Cadherin; FBS, fetal bovine serum; FGF, fibroblast growth factor; FGF1, fibroblast growth factor 1; FGF2, fibroblast growth factor 2; FITC-IB4, fluorescein labeled Griffonia Simplificata Isolectin B4; FITC, fluorescein; FOXD1, forkhead box 1; GATA3, GATA, binding protein 3; GDNF, glial cell derived neurotrophic factor; GGT, γ-glutamyl transferase; hESCs, human embryonic stem cells; hESCs, human pluripotent stem cells; hiPSCs, human induced pluripotent stem cells; HNF1β, hepatocyte nuclear factor-1, beta; HOXD11, homeobox D11; HPTCs, human primary proximal tubular cells; HSD3β, 3β-hydroxysteroid dehydrogenase; IM, intermediate mesoderm; ITGA8, integrin subunit alpha 8; IWR-1, tankyrase inhibitor; JAG1, jagged 1; KDR, kinase insert domain receptor; KSP, KSP-cadherin; KSR, knockout serum replacement; LDN193189, BMP, signalling inhibitor; LHX1, LIM, homeobox 1; LPS, late primitive streak; LTL, lotus tetragonolobus lectin; ME, mesoderm; MEIS1, meis homeobox 1; MET, mesenchymal–epithelial transition; MIXL1, Mix paired-like homeobox; MM, metanephric mesenchyme; N/A, not available; NCAD; N-Cadherin; NCAM, neural cell adhesion molecule; NPCs, nephron progenitor cells; OSR1, odd-skipped related transcription factor 1; PAN, puromycin aminonucleoside; PAX2, paired box 2; PAX8, paired box 8; PDGFRa2, Platelet Derived Growth Factor Receptor Alpha; PDGFRβ, Platelet-derived growth factor receptor beta; PIM, posterior primitive streak; PNA, peanut agglutinin; PODXL, podocalyxin; PS, primitive streak; PTH, parathyroid hormone; RA, retinoic acid; RhoA, ras homolog family member A; RV, renal vesicle; SALL1, spalt-like transcription factor 1; SB431542, TGFβ RI, Kinase inhibitor VI; scRNA-seq, Single cell RNA, sequencing; SIX1, SIX, homeobox 1; SIX2, SIX, homeobox 2; SLC3A1, solute carrier family 3 member 1; SOX9, SRY-Box transcription Factor 9; T, brachyury transcription factor; TEM, transmission electron microscopy; TUJ1, neuron-specific class III, beta-tubulin; UE, ureteric epithelia; UMOD, uromodulin; VEGFA, vascular endothelial growth factor A; VEGFR, vascular endothelial growth factor receptor; vWF, von willebrand factor; WD, wolffian duct precursors; WT1, Wilms tumor 1 gene; Y27632, ROCK, inhibitor.