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