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

Mature cells can be reprogrammed to regain their stem cell-like undifferentiated and proliferative state. These reprogrammed cells are called induced pluripotent stem cells or IPSCs. To differentiate pluripotent cells into podocytes-specialized cells involved in the filtration of waste from kidneys, begin by taking a suspension of human IPSCs in a mesoderm induction media.

Transfer this suspension onto a basement matrix coated culture plate and incubate. The basement matrix facilitates the adhesion of IPSCs at the base. Subsequently, the specific factors present in the media induce the differentiation of IPSCs into the mesoderm cells, the precursor cells for podocytes. Aspirate the spent media.

Supplement the plate with the desired volume of an intermediate mesoderm induction media and incubate for a prolonged duration. This step leads to the differentiation of mesodermal cells into immature podocytes. Replace the intermediate mesoderm induction media with a trypsin solution to dissociate the adherent cells from the plate.

Next, centrifuge to pelletize the cells. Discard the supernatant containing trypsin solution and matrix fragments. Resuspend the pellet in a podocyte induction medium. Transfer the cell suspension into a fresh coated culture plate. Incubate to allow the development of several foot-like projections from intermediate cells, resulting in the formation of mature podocytes.

After counting, resuspend the cells to a 1 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 1 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 to 15 of the differentiation, replace the mesoderm induction medium with 1 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 3 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 2 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 and podocyte induction medium at a 1 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 5 days.