Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Summary

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (>90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Abstract

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney’s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm’s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Introduction

Advances in human pluripotent stem cell culture are poised to revolutionize regenerative medicine, disease modeling, and drug screening by providing researchers with a renewable, scalable source of biological material that can be engineered to obtain almost any cell type within the human body¹. This strategy is especially useful for deriving specialized and functional cell types that would otherwise be difficult to obtain. Human induced pluripotent stem (hiPS) cells^2,3,4,5 are particularly attractive due to their soma....

Protocol

1. Preparation of reagents

1. 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.
2. Preparation of basement membrane (BM) matrix 1-coated plates for hiPS cell culture: Thaw BM matrix 1 overnight on ice at 4 ˚C. Once thawed, prepare aliquo.......

Discussion

In this report, we describe a protocol for the generation of kidney glomerular podocytes from hiPS cells. The hiPS cell-derived podocytes exhibit morphological and molecular features associated with the mature kidney podocyte phenotype¹³. In previous publications, we showed that the hiPS cell-derived podocytes can mimic the structure and selective filtration function of the kidney glomerulus when co-cultured with glomerular microvascular endothelial cells in a perfusable microfluidic organ-on-a-ch.......

Materials

Name	Company	Catalog Number	Comments
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 six-well plates	353046	Corning
VWR white techuni lab coat	10141-342	VWR
Wide-beveled cell lifter	3008	Corning