Deriving Human Kidney Podocytes from Induced Pluripotent Stem Cells: A Procedure for Directed Differentiation of Mature Kidney Podocytes from Stem Cells

Published: April 30, 2023

Abstract

Source: Burt, M. et al. Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions. J. Vis. Exp. (2020)

In this video, we describe the protocol to derive human kidney podocytes from induced pluripotent stem cells through cell differentiation under defined chemical conditions. The generated podocytes can be used for nephrotoxicity testing and disease modeling.

Protocol

1. Preparation of Reagents Dilute thawed 5x hiPS cell culture media (CCM) supplement in hiPS cell culture basal medium to obtain a 1x solution of hiPS CCM. NOTE: Frozen 5x hiPS CCM supplement requires a slow thawing process, ideally in 4 ˚C for overnight. Aliquots of the 1x hiPS CCM can be stored for up to 6 months at -20 ˚C. Preparation of basement membrane (BM) matrix 1-coated plates for hiPS cell culture: Thaw BM matrix 1 overnight on ice at 4 &#…

Divulgaciones

The authors have nothing to disclose.

Materials

Cells
DU11 human iPS cells The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.
Growth Factors and Media Supplements
All-trans retinoic acid (500 mg) 72262 Stem Cell Technologies
B27 serum-free supplement 17504044 Thermo/Life Technologies
CHIR99021 04-0004 Stemgent May show lot-to-lot variation
Complete Medium Kit with
CultureBoost-R
4Z0-500-R Cell Systems Podocyte maintenance media
DMEM/F12 12634028 Thermo/Life Technologies
DMEM/F12 with GlutaMAX supplement 10565042 Thermo/Life Technologies DMEM/F12 with glutamine supplement
Heat-inactivated FBS 10082147 Thermo/Life Technologies
Human activin A PHC9564 Thermo/Life Technologies
Human BMP7 PHC9544 Thermo/Life Technologies
Human VEGF PHC9394 Thermo/Life Technologies
mTeSR1 medium 05850 Stem Cell Technologies hiPS cell culture media (CCM)
Penicillin-streptomycin, liquid (100×) 15140-163 Thermo/Life Technologies
Y27632 ROCK inhibitor 1254 Tocris
Antibodies
Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies A32744; A32754; A-11076; A32790 Thermo/Life Technologies
Brachyury(T) ab20680 Abcam
Nephrin GP-N2 Progen
OCT4 AF1759 R&D Systems
PAX2 71-6000 Invitrogen
WT1 MAB4234 Millipore
ECM Molecules
iMatrix-511 Laminin-E8 (LM-E8) fragment N-892012 Iwai North America Basement membrane (BM) matrix 2
Matrigel hESC-qualified matrix, 5 mL vial 354277 BD Biosciences Basement membrane (BM) matrix 1. May show lot-to-lot variation
Enzymes and Other Reagents
Accutase A1110501 Thermo/Life Technologies Cell detachment solution
BSA A9418 Sigma-Aldrich
Dimethyl Sulfoxide (DMSO) D2438 Sigma-Aldrich DMSO is toxic. Should be handled in chemical safety hood
Enzyme-free cell dissociation buffer, Hank’s balanced salt 13150016 Thermo/Life Technologies
Ethanol solution, 70% (vol/vol), biotechnology grade 97065-058 VWR Ethanol is flammable and toxic
FBS 431097 Corning
Paraformaldehyde (PFA) 28906 Thermo/Life Technologies PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.
Phosphate-buffered saline (PBS) 14190-250 Thermo/Life Technologies
Sterile Distilled Water 15230162 Thermo/Life Technologies
Triton X-100 97062-208 VWR
Trypsin-EDTA, 0.05% 25300-120 Thermo/Life Technologies
Equipment
Aspirating pipettes, individually wrapped 29442-462 Corning
Avanti J-15R Centrifuge B99516 Beckman Coulter
Conical centrifuge tube, 15 mL 352097 Corning
Conical centrifuge tube, 50 mL 352098 Corning
Cryoboxes 3395465 Thermo/Life Technologies For storing frozen aliquots
EVOS M7000 AMF7000 Thermo/Life Technologies Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives
Hemocytometer 100503-092 VWR
Heracell VIOS 160i CO2 incubator 51030403 Thermo/Life Technologies For the routine culture and maintenace of iPS cells and their derivatives
Inverted Zeiss Axio Observer equipped with AxioCam 503 camera 491916-0001-000(microscope) ;
426558-0000-000(camera)
Carl Zeiss Microscopy Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation
Kimberly-Clark nitrile gloves 40101-346 VWR
Kimwipes, large 21905-049 VWR
Kimwipes, small 21905-026 VWR
P10 precision barrier pipette tips P1096-FR Denville Scientific
P100 barrier pipette tips P1125 Denville Scientific
P1000 barrier pipette tips P1126 Denville Scientific
P20 barrier pipette tips P1121 Denville Scientific
P200 barrier pipette tips P1122 Denville Scientific
Serological pipette, 10 mL, individually wrapped 356551 Corning
Serological pipette, 25 mL, individually wrapped 356525 Corning
Serological pipette, 5 mL, individually wrapped 356543 Corning
Steriflip, 0.22 μm, PES SCGP00525 EMD Millipore
Sterile Microcentrifuge Tubes 1138W14 Thomas Scientific For aliquoting growth factors
Tissue culture–treated 12-well plates 353043 Corning
Tissue culture–treated 6-well plates 353046 Corning
VWR white techuni lab coat 10141-342 VWR
Wide-beveled cell lifter 3008 Corning

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Deriving Human Kidney Podocytes from Induced Pluripotent Stem Cells: A Procedure for Directed Differentiation of Mature Kidney Podocytes from Stem Cells. J. Vis. Exp. (Pending Publication), e20557, doi: (2023).

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