We describe a protocol for generating human podocytes by episomal reprogramming dermal fibroblasts into human-induced pluripotent stem cells. These cells resemble in vivo podocytes much better than immortalized podocytes in terms of podocyte foot processes and expression of podocyte specific marker. Podocytes are terminally differentiated cells.
Therefore, these cells no longer proliferate and primary podocyte analysis in cell culture is limited. Even though the problem of limited cell number was overcome by using conditionally immortalized podocytes, these podocytes present a dedifferentiated phenotype and behavior, which questions their use in basic research. With the help of induced pluripotent stem cells, podocytes can be generated in vitro in an almost unlimited cell number with typical podocyte morphology, including distinct foot processes and expression of podocyte specific marker.
When using the patient's specific stem cells to differentiate into podocytes, it is possible to study the cells'disease-specific alterations ex vivo. In case, the initial skin biopsy was taken from a patient with a genetic podocyte disease. Podocytes generated with our protocol are personalized diseased cells carrying the patient's mutation.
Thus, these cells represent an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach. To begin, take the skin biopsy tube with fibroblast medium. Remove the medium and wash the skin punch biopsy thrice with five milliliters of sterile prewarmed PBS.
Using sterile forceps, transfer the skin punch biopsy to a 10-centimeter cell culture blade and cut it width-wise into three or four pieces with a sterile scalpel, leaving the epidermis and dermis. Transfer each piece into a sterile 35-millimeter plastic cell culture dish and press it gently into the culture dish. Allow it to dry for 5 to 10 minutes until the liquid evaporates and the biopsy is attached to the cell culture plastic.
Using a 1, 000-microliter pipette, add one milliliter of fibroblast medium dropwise around the biopsy to a final volume of three milliliters. Incubate the sample for seven days at 37 degrees Celsius and 5%carbon dioxide without feeding or moving the dish. At the end of the incubation, change the medium to fresh prewarmed fibroblast medium.
Finally, use a phase contrast microscope to monitor the outgrown spindle-like fibroblasts around the skin biopsy and freeze the selected cells after growing them. To begin, take a flask of cultured fibroblast cells. Code a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius.
Afterward, remove the media from the fibroblasts and wash them with four milliliters of prewarmed PBS per flask. Aspirate the PBS and dissociate the fibroblasts with four milliliters of trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius. Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface.
Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of prewarmed fibroblast medium. Collect and pool the remaining cells in the conical tube. Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius.
Aspirate the supernatant and resuspend the cell pellet in three milliliters of fresh prewarmed PBS. Count the cells. Transfer 1.5 times 10 to the 6 fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.
Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant. Resuspend the cell pellet in five milliliters of electroporation medium and centrifuge again.
In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of prewarmed fibroblast medium. At the end of the centrifugation, discard the supernatant and resuspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the 6 cells in 250 microliters. Transfer the 250-microliter cell suspension to an electroporation cuvette with a four millimeter gap distance.
Next, prepare a plasmid transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium. Transfer the transfection mix to the cuvette. Mix gently by flicking and electroporate with one pulse at 280 volts.
Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells and incubate overnight. Fibroblasts presented a long spindle-like phenotype with a size of 150 to 300 nanometers.
After reprogramming, colonies with 50 micrometer small hiPSCs occur, displaying distinct borders. The hiPSCs are distinguished by a high nuclei-to-body ratio and increased proliferation rate. To begin, take the plate of electroporated fibroblasts and observe the formation of hiPSC colonies under a 10x to 20x objective phase contrast microscope.
The hiPSC colonies with 300 micrometer diameter, distinct borders, and a high nuclei-to-body ratio are suitable for picking. Before picking, coat the wells of a 96-well plate suitable for hiPSC culture with 100 microliters of coating solution and incubate for one hour at 37 degrees Celsius. During incubation, mark the colonies of interest at the bottom of the cell culture dish with a pen.
For the final preparation of the 96-well plate, remove the coating solution and add 100 microliters of prewarmed hiPSC culture medium containing 10 micromoles of ROCK inhibitor Y27632 to each well. Next, wash the cells with prewarmed PBS and add fresh prewarmed hiPSC culture medium containing 10 micromoles of ROCK inhibitor Y27632 before picking to remove dead cells. Pick the hiPSC colonies using a gauge needle and divide the colonies into small pieces by drawing a grid into each colony.
Afterward, use a phase contrast microscope to check that the colonies are successfully divided into pieces. Finally, transfer the colonies with a 100-microliter pipette to the prepared 96-well plate, holding the pipette upright over the colony. Allow the cells to attach overnight at 37 degrees Celsius and 5%carbon dioxide without disturbance.
The following day, replace the medium with 200 microliters of fresh hiPSC culture medium to remove the ROCK inhibitor Y27632 and place the plates back in the incubator. Monitor the 96-well plate and mark the wells with successfully picked clones. The marked clones can be expanded and frozen at this point.
Phase contrast images have successfully reprogrammed hiPSC colonies, selected hiPSCs, spontaneously differentiated colonies, non-hiPSC colonies, and a too-dense hiPSC culture are presented.
