Primate IPSCs are useful but often difficult to obtain due to ethical and technical issues. This protocol gives a most accessible path to the generation and maintenance of primate IPSCs. The use of urinary cells as a primary source of IPSCs has a big advantage as they can be obtained in a completely non-invasive manner without any special techniques.
Demonstrating the procedure will be Jessica Radmer, a PhD student from Wolfgang M.S.Laboratory. To begin, centrifuge the urine containing tube at 400G for 10 minutes. Then carefully aspirate the supernatant, leaving approximately one milliliter in the tube and resuspend the pellet in it.
Wash the cells by adding 10 milliliters of urine wash buffer containing amphotericin and carefully mix the suspension using a serological pipette. After centrifugation, carefully aspirate the supernatant, leaving approximately less than 0.2 milliliters in the tube. Resuspend the cell pellet in one milliliter of primary urine medium containing amphotericin per 50 milliliters of initially processed urine.
Remove gelatin and add one milliliter of the suspension into a well of gelatin coated 12 well plate. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide. To prepare a basement membrane matrix coated 12 well plate, add 500 microliters of basement membrane matrix per well.
Move the plate to distribute the liquid and incubate it at 37 degrees Celsius for one hour. After incubation, replace the basement membrane matrix with 900 microliters of REMC medium and store it at 37 degrees Celsius until use. Calculate the volume of Sendai viruses needed for a multiplicity of infection of five using the equation.
Quickly thaw the components of the Sendai reprogramming kit in the water bath tempered at 37 degrees Celsius. Add REMC medium for a total of 100 microliters to a tube before adding the calculated volume of Sendai viruses and mixing. After dissociation of the urinary cells as described in the manuscript, count the cells using the cell counter and transfer the desired number of cells to a 1.5 milliliter tube.
Obtain the pellet by centrifugation and resuspend it in 100 microliters of the prepared Sendai virus mixture. Incubate the tube for one hour at 37 degrees Celsius for suspension infection. After incubation, add REMC to a total of one milliliter before seeding the cell suspension in the basement membrane matrix coated 12 well plate.
After transduction, change the medium every second day until the development of induced pluripotent stem cells, or IPSC, colonies with switching to PSC generation medium on day five. When the IPSC colonies grow large enough for clump passaging, aspirate the medium from the cultured cells and carefully wash the cells with 500 microliters of DPBS. After removing the DPBS, add 500 microliters of 0.5 millimolar EDTA to the well and incubate for two to five minutes.
Carefully observe the cells under the microscope until the colonies start detaching. When the edges of the colonies start to peel off and gaps between the cells become visible, remove the EDTA and carefully add 500 microliters of DPBS. Aspirate the DPBS and flush the well with 500 microliters of PSC culture medium using a P-1000 pipette.
Pipette up and down to disperse the colonies into clumps of appropriate size. Depending on the confluency, the desired density of the seeded cells and IPSC clonal preference, transfer 1/10 to 1/50 of the cell clump suspension to the new wells. Gently move the plate back and forth several times to distribute the clumps evenly in the well.
Incubate the plate for at least 30 minutes at 37 degrees Celsius to let the clumps attach. Change the medium every two to three days until the colonies grow large enough for passaging. After isolation, squamous cells and various smaller round cells can be seen that got excreted with the urine.
The first adherent proliferating cells can be seen, and these cells grow into large colonies that can be passaged. Two distinct cell types can be seen, one having an epithelial-like phenotype and the other showing a more mesenchymal-like phenotype with elongated shape. After the first passage, urinary cells grow as a monolayer.
During IPSC's generation, some adherent cells can be seen and these cells start to show morphological changes, indicating reprogramming of the cells. Further, the cells that form colonies display the typical embryonic stem cell morphology. All generated IPSCs share the typical embryonic stem cell-like colony morphology, characterized by tightly-packed cells with clearly-defined edges.
Immunocytochemistry was used to test the expression of pluripotency associated markers in gorilla IPSCs. Moreover, flow cytometry analysis showed that the analyzed orangutan IPSCs are positive for TRA-1-60. Furthermore, the gorilla IPSCs are able to differentiate into endoderm, mesoderm and ectoderm lineages.
An increased number of differentiated cells and a loss of border integrity and uniformity indicate poor IPSC quality. In contrast, defined borders, tight cellular packaging and prominent nucleoli signify high quality IPSCs. Due to the coronal variability, one needs to optimize the clone specific conditions such as splitting ratio, passage timing, and clump size to keep primate IPSCs growing healthy.
As quality controls of established IPSCs, one can verify pre-potency by direct or indirect differentiation induction such as in vitro EV differentiation, besides checking marker gene expression.
