Se requiere una suscripción a JoVE para ver este contenido. Inicie sesión o comience su prueba gratuita.
Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells
* These authors contributed equally
In This Article
Summary
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.
Abstract
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.
Introduction
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....
Protocol
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)
- Wash iPSCs on the membrane matrix-coated 6-well plate with 2 mL DPBS. Asp.......
Representative Results
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.......
Discussion
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.......
Acknowledgements
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).
....Materials
| 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  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 |
References
- Tekguc, M., et al. Kidney organoids: a pioneering model for kidney diseases. Translational Research. 250, 1-17 (2022).
- Hill, N. R., et al. Global prevalence of chronic kidney disease - a systematic review and met....
This article has been published
Video Coming Soon
ACERCA DE JoVE
Copyright © 2025 MyJoVE Corporation. Todos los derechos reservados