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 somatic cell origin and the potential they represent for personalized medicine. However, developing methods to derive other cell lineages from hiPS cells remain challenging due to the frequent use of poorly defined culture conditions which leads to low efficiency and non-specific generation of heterogenous cell populations^6,7.

Presented here is a method for the derivation of mature kidney podocytes from hiPS cells with specificity and high efficiency under chemically defined conditions. By considering the roles of multiple factors within the cellular microenvironment, a stem cell differentiation strategy was developed that involved the optimization of soluble factors presented in the cell culture medium as well as insoluble factors, such as extracellular matrix components or adhesive substrates. Given the importance of integrin signaling in podocyte development and function, the expression of integrin receptors on the cell surface was initially examined. β1 integrins were highly expressed not only in hiPS cells, but also in their derivatives including mesoderm and intermediate mesoderm cells^8,9,10. Subsequent experiments confirmed that ligands that bind to β1 integrins (including laminin 511 or laminin 511-E8 fragment) support the adhesion and differentiation of hiPS cells into podocytes when used in conjunction with the soluble inductive media described below.

Induction of cell lineage commitment was initiated by first confirming that hiPS cells cultured on the laminin-coated surfaces for two days in the presence of a medium containing Activin A, CHIR99021, and Y27632 Rock inhibitor can differentiate into cells that express the early mesoderm markers HAND1, goosecoid, and brachyury^8,11. Treatment of the mesoderm cells for 14 days with a medium supplemented with bone morphogenetic protein 7 (BMP-7) and CHIR99021 enabled the derivation of intermediate mesoderm cells that expressed the nephron-progenitor cell markers Wilm’s Tumor 1 (WT1), odd-skipped related protein 1 (OSR1)^8,11, and paired box gene 2 protein (PAX2)¹². To derive the mature kidney glomerular podocytes, the intermediate mesoderm cells were treated for 4–5 days with a novel medium consisting of BMP-7, Activin A, vascular endothelial growth factor (VEGF), all-trans retinoic acid, and CHIR99021. Flow cytometry and immunostaining were used to confirm that >90% of the resulting cells exhibited the molecular, morphological, and functional characteristics of the mature kidney podocyte^8,11,13. These characteristics include the development of primary and secondary foot processes; the expression of podocyte lineage-specific genes including SYNPO, PODXL, MAF, EFNB2⁸ and the expression of proteins including podocin, nephrin, and WT1^14,15,16. Additionally, it was found that the hiPS cell-derived podocytes can be maintained in culture for up to four weeks in vitro by using a commercially available medium^8,11 which provides an additional flexibility in the timing of downstream experiments. For more information regarding the flow cytometry panels used for determining the purity of the hiPS-podocytes, please refer to our previous publication¹¹.

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 aliquots with appropriate dilution factors as suggested by the manufacturer.
   NOTE: Typically, aliquots are prepared for subsequent dilution in 25 mL of cold DMEM/F12 in a 50 mL conical tube followed by thorough mixing to completely dissolve the BM matrix 1 and avoid the formation of residual crystals.
3. Transfer 1 mL of the BM matrix 1 solution to each well of a 6 well plate and incubate at 37 ˚C for 1-2 h or at 4 ˚C for a minimum of 24 h, wrapped in paraffin film. The BM matrix 1 -coated plates can be stored at 4 ˚C for up to 2 weeks.
4. Preparation of Basement membrane (BM) matrix 2 - coated plates: Dilute appropriate amounts of BM matrix 2 in 9 mL sterile distilled water to prepare a final concentration of 5 µg/mL. Add 700 µL of the BM matrix 2 solution to each well of a 12 well plate and incubate the plate at room temperature for 2 h or overnight at 4 ˚C.
5. Prepare 100 µg/mL stock solutions each of BMP7, Activin A, and VEGF as follows: reconstitute BMP7 in sterile distilled water containing 0.1% (wt/vol) BSA and reconstitute Activin A and VEGF separately in sterile PBS containing 0.1% (wt/vol) BSA. To avoid frequent freeze-thaw cycles, prepare 100 µL aliquots from each stock solution and store at -20 ˚C for up to 6 months.
6. Prepare a 10 mM stock solution of Y27632 by dissolving 10 mg of Y27632 in 3.079 mL of sterile distilled water. Aliquot 100 µL from the stock and store at -20 ˚C for up to 6 months.
7. Dissolve 2 mg of CHIR99021 in 143.4 µL of sterile DMSO to prepare a 30 mM stock solution. Prepare 5 µL aliquots and store at -20 ˚C for up to 1 month (or according to the manufacturer’s recommendation).
8. Dissolve 10 mg of all-trans retinoic acid in 3.33 mL sterile DMSO. Prepare 500 µL aliquots and store at -20 ˚C for up to 6 months.

2. Preparation of culture media

1. Prepare the mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL Activin A, 3 µM CHIR99021, 10 µM Y27632, and 1x B27 serum-free supplement in an appropriate volume of DMEM/12 with glutamine supplement.
   NOTE: The mesoderm differentiation medium should be freshly prepared before differentiation steps and at a volume appropriate for the scale of the experiment (typically, 50 mL of medium is adequate for two 12 well plates).
2. Prepare intermediate mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL BMP7, 3 µM CHIR99021, and 1x B27 serum-free supplement in DMEM/F12 with a glutamine supplement.
   NOTE: If needed, the medium can be supplemented with 1% (vol/vol) Penicillin-Streptomycin. Adjust the volume of the medium by using DMEM/F12 with glutamine supplement. This medium can be prepared in large batches; however, it is recommended to store in smaller aliquots (e.g., 45 mL in 50 mL conical tubes) to avoid repeated freeze-thaw cycles. This media can be stored at -20 ˚C for up to 3 months and can be thawed at 4 ˚C overnight prior to use.
3. Prepare the podocyte induction medium by reconstituting to a final concentration 100 ng/mL BMP7, 100 ng/mL activin A, 50 ng/mL VEGF, 3 µM CHIR99021, 1x B27 serum-free supplement, and 0.1 µM all-trans retinoic acid in DMEM/F12 with glutamine supplement. Protect medium from light (e.g., by wrapping container with foil paper).
   NOTE: This medium can be prepared in large batches and stored in the dark at -20 ˚C for up to 3 months. Frozen aliquots should be thawed overnight at 4 ˚C prior to use.
4. Prepare 25 mL of trypsin neutralizing solution by adding 10% (vol/vol) heat inactivated FBS in DMEM/F12 and filter under sterile conditions.
5. For post-differentiation maintenance of the stem cell-derived podocytes, prepare Complete Medium with podocyte maintenance media by adding the supplement to the basal medium as per the manufacturer’s guidelines, and store at 4 ˚C for up to two weeks.

3. Feeder-free hiPS cell culture using hiPS cell culture medium

1. Aspirate the residual solution of BM matrix 1 from the pre-coated plates and wash the wells 3 times with 1 to 2 mL of warmed DMEM/F12.
2. Aspirate spent hiPS CCM from the hiPS cells and rinse the cells 3 times with warmed DMEM/F12. Add 1 mL of warm cell detachment solution and incubate for 1 min at 37 ˚C to help dissociate the cells. Perform visual inspection of cells under a tissue culture microscope and ensure that the edges of the cell colonies appear rounded, then quickly aspirate the cell detachment solution from the cells (ensuring that the cell colonies are still attached to the plate, albeit loosely). Gently rinse cells once with DMEM/F12 to remove the cell detachment solution.
3. Add 3 mL of hiPS CCM to the hiPS cells (in each well of a 6-well plate) that were treated with the cell detachment solution. Scrape colonies by using a cell lifter, and gently pipette cell suspension up and down to dislodge the loosely adhered cells. Wash the plate thoroughly to ensure all the cells are harvested.
   NOTE: Use of a 5 mL pipette is recommended to avoid excess shear on the cells.
4. Transfer 0.5 mL of the cell suspension into each well of a new BM matrix 1-coated 6-well plate containing 2 mL of hiPS CCM per well. Move the plate in figure-eight fashion to distribute cell colonies within wells and incubate at 37 ˚C in a 5 % CO₂ incubator. Refresh medium daily until the cells are ready to be passaged or used for the differentiation experiment (approximately 70 % confluency).
   NOTE: The ideal colony size for routine passaging of iPSCs is between 200-500 µm under feeder free conditions using hiPS CCM (without ROCK inhibitor). If cells are individualized during treatment with dissociation enzymes or buffers, the hiPS CCM can be supplemented with ROCK inhibitor (e.g., 10 µM Y27632) to improve cell viability.

4. Differentiation of hiPS cells into mesoderm cells (days 0-2)

1. While the hiPS cell cultures are in the exponential growth phase (approximately within 4 days of culture after passaging, and around 70% confluency), visually inspect for the presence of spontaneously differentiated cells within and around the edges of the colonies. If necessary, aseptically scrape-off areas of differentiation.
2. Aspirate hiPS CCM from hiPS cells and rinse the cells 3 times with warm DMEM/F12. Incubate the cells with 1 mL of enzyme-free cell dissociation buffer for 10 min at 37 ˚C and check for the dissociation under a microscope. Due to inherent differences between different hiPS cell lines, the actual incubation time for the cell dissociation buffer must be determined for a given cell line.
3. Gently scrape the well with a cell lifter to dislodge loosely adhered cells and transfer the cell suspension to a 15 mL conical tube, followed by pipetting up and down several times to individualize the hiPS cells.
   1. Bring cell suspension to the 15 mL volume with warm DMEM/F12 and centrifuge for 5 min at 290 x g at room temperature.
   2. Gently aspirate the supernatant and resuspend the cells with warm DMEM/F12 for another round of centrifugation to remove residual BM matrix 1 and dissociation buffer components.
4. Aspirate the supernatant and resuspend cells in 1 mL of mesoderm induction medium as described in above. Count the total number of cells using a hemocytometer or coulter counter to determine the appropriate volume of mesoderm differentiation medium necessary to achieve a concentration of 1 x 10⁵ cells/mL.
5. Aspirate ECM solution from the BM matrix 2-coated plates and rinse the plates twice with warm DMEM/F12. Mix the hiPS cell suspension gently by pipetting a few times. Transfer 1 mL of the cell suspension to each well of the BM matrix 2-coated 12-well plates and then gently shake the plates to distribute the cells more evenly.
6. Incubate the plate at 37 ˚C in a 5% CO₂ incubator. Refresh the mesoderm induction medium the next day.
   NOTE: After 2 days, hiPS cell-derived mesoderm cells would be ready for intermediate mesoderm induction.

5. Differentiation of hiPS cell-derived mesoderm cells into intermediate mesoderm (days 2-16)

1. On the day 2 of the differentiation protocol, aspirate mesoderm induction medium and replenish with 1 mL per well intermediate mesoderm induction medium.
2. Refresh medium every day to maintain an accurate threshold of growth factors and small molecules for the metabolically active cells. If there is substantial cell growth and rapid depletion of media nutrients (indicated by the yellowing of the media), the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 mL per well of the 12 well plates.
3. Culture cells for additional 14 days to obtain intermediate mesoderm cells. By day 16, these cells can be cryopreserved for later use.

6. Differentiation of hiPS cell-derived intermediate mesoderm cells into podocytes (days 16 to 21)

1. Rinse the intermediate mesoderm cells with warm DMEM/F12 followed by incubation of the cells with 0.5 mL per well of 0.05 % trypsin-EDTA for 3 min at 37 ˚C. Perform visual inspection to ensure that the cells are beginning to dissociate.
2. Scrape cells using a cell lifter and pipette the cell suspension several times using a 1,000 µL pipette tip to obtain individualized (or small clumps of) cells.
   NOTE: At this stage, ensure that the cells are fully dissociated as aggregated cells may fail to acquire a terminally differentiated phenotype within the timeline of the protocol.
3. Add about 2 mL per well of trypsin neutralizing solution to stop the activity of trypsin.
4. Transfer cells to a 50 mL conical tube and bring the volume up to 50 mL using DMEM/F12, and then centrifuge the cell suspension for 5 min at 201 x g at room temperature.
5. Aspirate the supernatant and resuspend cells in the podocyte induction medium. For optimal results, ensure a final seeding density of approximately 100,000 cells/well of a 12 well plate. Add the cell suspension to the BM matrix 2-coated plates and gently shake the plate to help distribute the cells more evenly.
6. Incubate cells at 37 ˚C and 5% CO₂ and refresh medium daily for up to 5 days to obtain podocytes by day 21.
   NOTE: The resulting hiPS cell-derived podocytes can be maintained in culture for 2-4 additional weeks by using complete medium with podocyte maintenance media. Once in podocyte maintenance media, the cells can be fed every other day and may be used for subsequent studies or downstream analyses.

Results

The goal of this protocol was to demonstrate that mature human podocytes can be derived from hiPS cells under chemically defined conditions. The data presented in this manuscript were generated by using the DU11 hiPS cell line¹⁷, which were first tested for, and found to be free of mycoplasma. Chromosomal analysis was also performed, and the cells were found to be karyotypically normal. Starting with the undifferentiated DU11 hiPS cells, the differentiation strategy (Figure 1<...

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

List of materials used in this article

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