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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Published: September 1st, 2023

DOI:

10.3791/65698

1Department of Nephrology, The Children's Hospital, Zhejiang University School of Medicine, 2Zhejiang University School of Medicine, 3Hubei Normal University, 4National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, 5Clinical Laboratory, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health
* These authors contributed equally

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

The kidney plays a critical role in maintaining physiological homeostasis, depending on its functional unit. Nephrons, which excrete waste products, can regulate the composition of body fluids. Chronic kidney disease (CKD), caused by hereditary mutations or other high-risk factors, will eventually progress to end-stage kidney disease (ESKD)1,2. ESKD is apparently due to the limited regeneration capacity of nephrons. Thus, renal replacement therapy is required. Directed differentiation of human iPSCs enables the in vitro generation of patient-specific 3D kidney organoids, which can be used to study kid....

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The iPSCs used for the present study were obtained from a commercial source. The cells were maintained with mTeSR medium on commercially available basement membrane matrix-coated plates (see Table of Materials). Table 1 contains all the medium compositions utilized in the study.

1. Plating iPSCs for differentiation and inducing posterior primitive streak (PS)

  1. Wash iPSCs on the membrane matrix-coated 6-well plate with 2 mL DPBS. Asp.......

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The production of IM is achieved by activating canonical WNT signaling using the GSK3 inhibitor CHIR99021, followed by FGF9/heparin. From Day 0 to Day 4, iPSCs rapidly expand and take on rhomboid or triangular shapes. The confluence reaches 90%-100% and accumulates evenly until Day 7. Upon suspension culture, the aggregates spontaneously form nephron structures after dissociating on Day 7. The kidney organoids created through suspension culture display tubular-like structures and are easily observed in bright field image.......

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A detailed protocol has been described for generating kidney organoids from iPSCs, involving minor modifications to the basal medium, initial cell density, and concentration of CHIR99021. In various experiments, the critical factors for successful kidney organoid generation were found to be the initial differentiation of the intermediate mesoderm (IM) and the cell state on Day 7. Moreover, different iPSC lines exhibited variations in cell proliferation and differentiation potential, resulting in varying optimal cell dens.......

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We are extremely grateful to all Mao and Hu Lab members, past and present, for the interesting discussions and great contributions to the project. We thank the National Clinical Research Center for Child Health for the great support. This study was financially supported by the National Natural Science Foundation of China (U20A20351 to Jianhua Mao, 82200784 to Lidan Hu), the Natural Science Foundation of Zhejiang Province of China (No. LQ22C070004 to Lidan Hu), and the Natural Science Foundation of Jiangsu Province (Grants No. BK20210150 to Gang Wang).

....

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Name Company Catalog Number Comments
96 Well Cell Culture Plate, Flat-Bottom NEST Cat #701003
Accutase STEMCELL Technologies Cat #o7920
Antibodies
Benzyl alcohol Sigma-Aldrich Cat #100-51-6
Benzyl benzoate Sigma-Aldrich Cat #120-51-4
Biological Safety Cabinet Haier Cat #HR40 Equation 1 A2
Biotin anti-human LTL (1:300) Vector Laboratories Cat #B-1325
Blood mononuclear cells hiPS-B1 (iPSc, female) N/A N/A
Carbon dioxide level shaker HAMANY Cat #C0-06UC6
Chemicals, peptides, and recombinant proteins
CHIR99021 (Wnt pathway activator) STEMCELL Technologies Cat #72054
Costar Multiple 6 Well Cell Culture Plate Corning Cat #3516
Costar Ultra-Low Attachment 6 Well Plate Corning Cat #3471
CryoStor CS10 STEMCELL Technologies Cat #07930
DAPI stain Solution Coolaber Cat #SL7102
Dextran, Alexa Fluor 647 Thermo SCIENTIFIC Cat #D22914
DMEM/F-12 HEPES-free Servicebio Cat #G4610
Donkey Anti-Sheep IgG H&L (Alexa Fluor 647) Abcam Cat #ab150179
Donkey serum stoste Meilunbio Cat #MB4516-1
D-PBS (without calcium, magnesium, phenol red) Solarbio Life Science Cat #D1040
Dry Bath Incubator Shanghai Jingxin Cat #JX-10
Dylight 488-Goat Anti-Mouse IgG (1:400) Earthox Cat #E032210
Dylight 488-Goat Anti-Rabbit IgG (1:400) Earthox Cat #E032220
Dylight 549-Goat Anti-Mouse IgG (1:400) Earthox Cat #E032310
Dylight 549-Goat Anti-Rabbit IgG (1:400) Earthox Cat #E032320
Dylight 649-Goat Anti-Rabbit IgG (1:400) Earthox Cat #E032620
Experimental models: Cell Lines
Forma Steri-Cycle CO2 Incubator Thermo SCIENTIFIC Cat #370
Geltre LDEV-Free Gibco Cat #A1413202
Glass Bottom Culture Dishes NEST Cat #801002
Goat anti-human CUBN (1:300) Santa Cruz Biotechnology Cat #sc-20607
Heparin Solution (Cell culture supplement) STEMCELL Technologies Cat #07980
Human Recombinant FGF-9 STEMCELL Technologies Cat #78161
Inverted Microscope OLYMPUS Cat #CKX53
Laser Scanning Confocal Microscope OLYMPUS Cat #FV3000
Methyl cellulose Sigma-Aldrich Cat #M7027
Micro Centrifuge HENGNUO Cat #2-4B
Mouse anti-human CD31 (1:300) BD Biosciences Cat #555444
Mouse anti-human ECAD (1:300) BD Biosciences Cat #610182
Mouse anti-human Integrin beta 1 (1:300) Abcam Cat #ab30394
Mouse anti-human MEIS 1/2/3 (1:300) Thermo SCIENTIFIC Cat #39795
Mowiol 4-88 (Polyvinylalcohol 4-88) Sigma-Aldrich Cat #81381
mTeSR1 5X Supplement STEMCELL Technologies Cat #85852
mTeSR1 Basal Medium STEMCELL Technologies Cat #85851
Nunc CryoTube Vials Thermo SCIENTIFIC Cat #377267
Others
Rabbit anti-human GATA3 (1:300) Cell Signaling Technology Cat #5852S
Rabbit anti-human LRP2 (1:300) Sapphire Bioscience Cat #NBP2-39033
Rabbit anti-human Synaptopodin (1:300) Abcam Cat #ab224491
Rabbit anti-human WT1 (1:300) Abcam Cat #ab89901
Rabbit anti-mouse PDGFR (1:300) Abcam Cat #ab32570
Recombinant Human Serum Albumin (rHSA) YEASEN Cat #20901ES03
Sheep anti-human NPHS1 (1:300) R&D Systems Cat #AF4269
STEMdiff APEL 2 Medium STEMCELL Technologies Cat #05275
Streptavidin Cy3 (1:400) Gene Tex Cat #GTX85902
Versene (1X) Gibco Cat #15040066
Y-27632 (Dihydrochloride) STEMCELL Technologies Cat #72304

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