Protocols for the differentiation of stem cells into functional kidney cells remain elusive. This method produces mature podocytes with specificity and efficiency, providing new tools for studying kidney disease mechanisms. This method employs cell culture medium and extracellular matrix proteins with a chemically-defined composition to produce human iPS cell derived podocytes with a high purity without using subpopulation selection or genetic manipulation.
There are limited therapeutic options for patients with kidney disease. This method has implications for modeling and understanding disease mechanisms, which could facilitate the development of novel biomarkers and therapeutics. To set up a feeder-free human iPSC culture, aspirate the residual solution of basement membrane matrix 1 from pre-coated plates and wash the wells three times with one to two milliliters per well of warmed basal medium.
Aspirate the spent cell culture medium from a human iPSC culture and rinse the cells three times with warmed medium. Add one milliliter of warm cell detachment solution to each well for a one-minute incubation at 37 degrees Celsius. After confirming rounded cell colony edges under a tissue culture microscope, quickly aspirate the cell detachment solution and gently rinse the cells with cell culture medium.
Add three milliliters of human iPSC culture medium to the human-induced pluripotent stem cells and use a cell lifter to scrape the colonies. Gently pipette the cell suspension to dislodge any loosely adhered cells and wash the plate thoroughly to ensure harvesting of all of the cells. Transfer 500 microliters of cells into each well of a new basement membrane matrix 1 coated six-well plate containing two milliliters of human-induced pluripotent stem cells per well and move the plate in a figure eight fashion to evenly distribute the cell colonies within the wells.
Place the plate in the cell culture incubator, refreshing the medium daily until the cells reach approximately 70%confluency. If necessary, aseptically scrape off areas of differentiation before aspirating the supernatant from the wells. After washing the wells three times, dissociate the cells as demonstrated and pull the resulting cell suspensions in a 15 milliliter conical tube.
After mixing by pipetting, bring the final volume in the tube to 15 milliliters with warm medium and collect the cells by centrifugation. Resuspend the pellet in fresh medium for a second centrifugation and resuspend the cells in one milliliter of mesoderm induction medium for counting. After counting, resuspend the cells to a one times 10 to the fifth cells per milliliter of mesoderm induction medium concentration and aspirate the extracellular matrix solution from the basement membrane matrix 2 coated plates.
Rinse the plates two times with warm medium and mix the human-induced pluripotent stem cell suspension with gentle pipetting. Add one milliliter of cells to each well of the basement membrane matrix 2 coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then place the plate into the cell culture incubator.
On days 2-15 of the differentiation, replace the mesoderm induction medium with one milliliter of intermediate mesoderm induction medium per well. If substantial cell growth and a rapid depletion of nutrients is observed as indicated by yellowing of the medium, the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 milliliters per well. On day 16 of culture, rinse the intermediate mesoderm cells with warm medium and incubate the cells with 500 microliters of 0.05%Trypsin EDTA per well for three minutes at 37 degrees Celsius.
When the cells begin to dissociate, scrape the cells with a cell lifter and gently mix the cells by pipetting. Stop the reaction with about two milliliters of Trypsin neutralizing solution per well and transfer the cells to a 50 milliliter conical tube. Bring the volume up to 50 milliliters with medium and collect the cells by centrifugation.
Resuspend the pellet in podocyte induction medium at a one times 10 to the fifth cells per milliliter of medium concentration and add the cells to basement membrane matrix 2 coated plates. Then gently shake the plate to help distribute the cells more evenly and place the cells into the incubator for up to five days. Using the culture strategy as demonstrated, the stem cells can be first differentiated into mesoderm cells that express Brachyury, followed by differentiation into PAX2 positive intermediate mesoderm cells, and eventually mature kidney glomerular podocytes.
Stem cell derived podocytes stain positive for WT1, as well as for the lineage identification marker nephrin. Intriguingly, the subcellular localization of nephrin is predominantly within the podocyte foot processes and cell cytoplasm, consistent with a mature podocyte phenotype. Cells seeded at densities that significantly exceed the recommended density of one times 10 to the fifth cells per well of a 12-well plate result in large clusters of cells that lack the expected morphological phenotype of mature podocytes within the standard timeline of the protocol.
Seeding at the densities recommended in this protocol, however, results in cultures of cells with the desired morphology. We recommend using the appropriate basement membrane protein for cell detachment and maintaining the correct cell seeding density for mesoderm and podocyte induction. This podocyte differentiation method can be integrated with organs-on-chips microfluidic systems or 3D bioprinting technologies to develop functional models of the human kidney for drug screening and nephrotoxicity testing.
The lack of appropriate cell culture models has impeded progress in understanding kidney disease mechanisms. This protocol provides researchers with an inexhaustible source of patient-derived podocytes for disease modeling.
