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Abstract

Introduction

Protocol

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Materials

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Developmental Biology

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Published: April 13th, 2021

DOI:

10.3791/62452

1Department of Developmental Biology, University of Pittsburgh, School of Medicine, 2Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 3Center for Critical Care Nephrology, University of Pittsburgh, School of Medicine

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

In recent years, a number of protocols to differentiate human pluripotent stem cells into kidney organoids have been developed1,2,3,4,5. Kidney organoids have provided an important tool to aid research into new regenerative medicine approaches, model kidney-related diseases, perform toxicity studies and therapeutic drug development. Despite their wide applicability, kidney organoids have limitations such as lack of maturation, limited long-term culture capacity in vitro, and a paucity of several ce....

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All experiments using hPSCs were performed in compliance with institutional guidelines, and were carried out in a Class II biosafety hood with appropriate personal protective equipment. All reagents are cell culture-grade unless stated otherwise. All cultures are incubated at 37 °C, 5% CO2 air atmosphere. At all stages of the assay, embryoid bodies or kidney organoids can be collected, and fixed or prepared for analysis. The hPSC lines used to generate this data have been fully characterized and published.......

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In this most recent version of our protocol, kidney organoid differentiation is initiated in a defined, low protein medium. The assays are performed entirely in suspension and rely on the innate ability of hPSCs differentiation and organization for initiation of tubulogenesis. A single assay originating from a 100 mm ~60% confluent hPSC culture plate routinely yields 500-1,000 kidney organoids, as shown in our previous publication5. Due to such high numbers of organoids generated, this protocol is.......

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Previous studies have shown that the initial protocol steps are critical for intermediate mesoderm differentiation5,19,20 and, therefore, it is essential to implement a stringent medium composition at this stage. Removing undefined components such as serum, albumin, protein free hybridoma medium II from the first stage of the protocol may help to improve consistent differentiation efficiency between assays21

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This research was funded by the National Institutes of Health R01 DK069403, UC2 DK126122 and P30-DK079307 and ASN Foundation for Kidney Research Ben J. Lipps Research Fellowship Program to AP.

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Name Company Catalog Number Comments
2-Mercaptoethanol Thermo Fisher 21-985-023
Anti-adherence rinsing solution STEMCELL Technologies 7010
CHIR99021 STEMCELL Technologies 72054 10 mM stock in DMSO
Corning disposable spinner flasks Fisher Scientific 07-201-152
Corning Ultra-Low Attachment 6-well plates Fisher Scientific 07-200-601
Corning Slow-Speed Stirrers Fisher Scientific 11-495-03 Multi plate magnetic stirrer for spinner flask culture
Dispase STEMCELL Technologies 7923 Aliquot and freeze
DMEM, low glucose, pyruvate, no glutamine, no phenol red Thermo Fisher 11054020
DPBS 1x, no calcium, no magnesium Thermo Fisher 14-190-250
Egg / Oval Stirring Bars 2mag PI20106
Excelta General-Purpose Tweezers Fisher Scientific 17-456-103 Keep sterile in the cell culture hood
EZBio Single Use Media Bottle, 250mL Foxx Life Sciences 138-3211-FLS Used to make PVA 10%
Falcon Standard Tissue Culture Dishes (100 mm) Thermo Fisher 08-772E
Fisherbrand Sterile Aspirating Pipet 2mL Fisher Scientific 14-955-135
Fisherbrand  Cell Lifters - Cell lifter Fisher Scientific 08-100-240
Fisherbrand Multi Function 3D Rotators Fisher Scientific 88-861-047 Orbital shaker
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix Thermo Fisher A1413302 BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex.
Gentle Cell Dissociation Reagent STEMCELL Technologies 7174 GCDR
GlutaMAX Supplement Thermo Fisher 35-050-061 L-glutamine supplement.
HEPES (1M) Thermo Fisher 15-630-080
Insulin-Transferrin-Selenium-Ethanolamine Thermo Fisher 51-500-056 ITSE
KnockOut  Serum Replacement - Multi-Species Thermo Fisher A3181502 KOSR. Aliquot and freeze
Lipid Mixture 1, Chemically Defined Millipore-Sigma L0288-100ML
MEM Non-Essential Amino Acids Solution Thermo Fisher 11140-050
MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL Fisher Scientific S2GPU05RE
MilliporeSigma  Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL Fisher Scientific S2GPU02RE
MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit 2mag VMF 90250 U
MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive 2mag VMF 40600 6MSP
MP Biomedicals  7X Cleaning Solution Fisher Scientific MP0976670A4 Tissue culture suitable detergent. Make a 5% solution in water
mTeSR1 STEMCELL Technologies 85850 hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives. 
Nunc 50 mL Conical, Sterile Centrifuge Tubes Fisher Scientific 12-565-270
Nunc 15mL Conical Sterile Centrifuge Tubes Fisher Scientific 12-565-268
Penicillin-Streptomycin Thermo Fisher 15-140-122 Aliquot and freeze
Plasmocin Invivogen ant-mpt Anti-mycoplasma reagent. Aliquot and freeze
pluriStrainer® 200 µm Fisher Scientific NC0776417 Cell strainer
pluriStrainer® 500 µm Fisher Scientific NC0822591 Cell strainer
Poly(vinyl alcohol) 87-90% hydrolyzed  (PVA) Millipore-Sigma P8136-250G 10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS
ROCK inhibitor Y-27632 (ROCKi) STEMCELL Technologies 72304 10 mM stock in DPBS
Sterile Disposable Serological Pipets  - 10mL Fisher Scientific 13-678-11E
Sterile Disposable Serological Pipets - 25mL Fisher Scientific 13-678-11
Sterile Disposable Serological pipette - 5 mL Fisher Scientific 13-678-12D
TeSR-E5 STEMCELL Technologies 5916 Serum-free, low protein base medium for E5-ILP
Variomag distriBOX 2 Distributor 2mag VMF 90512 If you use more than one MIXdrive

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