This method can help answer key questions in the human germline toxicology field, such as how damages in the genome of human primordial germ cells are created or repaired. The main advantage of this technique is that human primordial germ cells can be generated in two weeks from the primed pluripotency iPS cells harboring disease predisposition genetic background. The implications of this technique extend toward prevention of genomic impairment in human germline cells, because our cell culture model can help us to reduce germline toxicity of a therapeutic drug.
To begin with generation of human primordial germ cell-like cells, or human PGCLCs, prepare extracellular matrix protein-coated plates and cell suspension of human iPSCs, as described in the text protocol. Prepare a single-cell suspension of primed pluripotency human iPSCs in the mTeSR1 medium containing the Y-27632 Rho kinase inhibitor. Carefully adjust the cell density to 100, 000 cells per milliliter.
Inoculate two milliliters of cell suspension of primed human iPSCs in each well of the extracellular matrix protein coated six-well plates, making sure that the cells are evenly distributed by shaking the plates vertically and horizontally. Incubate the plates in a tri-gas CO2 incubator at 37 degrees Celsius. It is very important to inoculate exactly 200, 000 cells in each well of a six-well plate.
Accurate counting of cells is critical. After completion of the transient conversion to naive pluripotency, cells should look over-confluent, which is normal. Exact timing of medium change is important to achieve high human PGCLCs and experimental reproducibility.
Density of the 4i hiPSC culture is high under these conditions, and it is expected for cell to become confluent at the 48 to 72 hours after inoculation. 24 hours after inoculation, change the medium with two milliliter per well of mTeSR1 medium without Y27632. Start conversion of primed pluripotency state human iPSCs to 4i naive pluripotent stem cells, by warming 50 milliliter of 4i complete medium in a 37 degrees Celsius water bath for 15 minutes.
At 48 and 72 hours after inoculation, remove the plates from the incubator, and change the medium with 2 milliliters per well of 4i complete medium. Centrifuge the detergent coated microwell plate at 1, 000 g for five minutes at room temperature. Inspect the wells with inverted microscope to ensure the absence of air bubbles.
Rinse the wells as described in the text protocol, and repeat the centrifugation. Add 4i complete medium to the rinsed wells. Centrifuge the plate at 1, 000 g for five minutes at room temperature, and inspect the wells again.
To inoculate 4i human iPSCs into the microwell plate, prepare human iPSC cell suspension as described in the protocol. Filter this cell suspension through a 40 micrometer pore size cell strainer to remove cell aggregates. Wash the strainer with one milliliter of pre-warmed 4i complete medium three times to collect all the cells from the strainer, and count the cells with a culture counter.
Then, dilute this single cell suspension of 4i naive human iPSCs to 27 to 32.4 million cells in 9 milliliters pre-warmed 4i complete medium containing Y27632. Remove previously prepared microwell plate containing 1 milliliter of 4i complete medium per well from a tri-gas incubator. Inoculate 1 milliliter of 4i naive human iPSC suspension into each well, to achieve a concentration of 3.0 to 3.6 million cells per well.
Gently pipette to evenly distribute the cells. Centrifuge the plate at 100 g for 3 minutes at room temperature. Place the plate in a tri-gas CO2 incubator, making sure not to disturb cells pelleted in microwells, and incubate for 24 to 30 hours, because an overnight incubation is insufficient for formation of tight embryonic bodies or EBs.
Prewarm 5 milliliters of human PGCLC basal medium and 20 milliliters of human PGCLC complete medium for at least five minutes in a 37 degrees Celsius water bath Carefully place a 40 micrometer pore sized cell strainer upside down over a 50 milliliter polypropylene conical tube. To detach EBs from microwells, gently pipette each well of the plate. To remove cells not incorporated in EBs, filter the contents of all wells through the cell strainer.
Gently wash the EBs retained on the membrane of the cell strainer with 1 milliliter of pre-warmed human PGCLC basal medium, and repeat the wash five times. Place a fresh 50 milliliter conical tube on the cell strainer, so that the cell strainer is now positioned right side up in the new tube. Quickly invert the cell strainer with the new tube, which will position the EBs below the membrane of the cell strainer.
Add 18 milliliters of pre-warmed human PGCLC complete medium to the membrane of the cell strainer to collect EBs in the conical tube. Then, plate 3 milliliters per well of the suspension of EBs into wells of a low attachment six-well plate. Place the plate on a seesaw-move rocker in a tri-gas incubator, and carefully set the rocking speed at approximately 20 turns per minute.
On day seven to 13 of the experiment, freshly prepare 20 milliliters of human PGCLC complete medium each day. Remove the plate from the rocker, which will make the EBs sink to the bottom of the wells. Remove the old medium without drying EBs so that about 0.2 milliliters of old medium remains in each well.
Add 3 milliliters per well of fresh human PGCLC complete medium, after removing the old medium everyday. To identify human PGCLCs as OCT4-positive cells, harvest the EBs to a 1.5 milliliter low-bind microcentrifuge tube. Let the EBs sink to the bottom at room temperature, and discard the medium.
Rinse the EBs with ice cold 1X PBS. After removing the PBS, incubate the EBs in 1 milliliter of ice cold 1%weight by volume sodium azide in PBS on ice for five minutes. Let the EBs sink to the bottom at room temperature.
After discarding the supernatant, rinse the EBs with ice cold PBS. Thaw 200 microliters of extracellular matrix protein on ice. Add the protein to the tube containing EBs.
Leave the tube at room temperature for approximately 10 minutes, until the gel is solidified, and the EBs are embedded. To fix the EBs, add 1 milliliter of 4%formaldehyde in PBS to the tube, and incubate at room temperature for 15 minutes with gentle rocking. After that, discard the formaldehyde and rinse the EBs once with ice cold PBS.
Add 1 milliliter of 70%ethanol. Store in 70%ethanol at 4 degrees Celsius for up to one week, or proceed to process them with a standard FFPE protocol. Cell density and even distribution of cells on each well are critical.
Human IPSCs in 2 milliliters of Y27632 supplemented medium per well of a six-well plate, reach 20 to 30%confluency after 24 hours. After an additional 24 hour culture in the mTeSR1 medium, in the absence of Y27632, cells aggregated and formed colonies, occupying approximately 30%of the growth area. After 24 hours of culture in the 4i reprogramming medium, cells reached confluency.
After an additional 24 hours of culture in the 4i medium, cells became more densely packed. After a 24 hour incubation in microwells in the 4i reprogramming medium, cells formed EBs with their circular contour visible at 24 to 30 hours after inoculation. EBs kept in the human PGCLC medium, under rocking nonadherent conditions, maintained their spherical shape without aggregation after 24 hours.
After 192 hours, EBs were larger, maintaining the same morphology. After 5 days, the cells emerged as OCT4 expressing cells on the surface of EBs, increasing their number after 8 days. Cells from single cell suspension of human PGCLCs were enriched as CD38-positive cells by FACS.
Cells that do not express CD38 were negative control. To avoid contamination, FACS gates CD38-positive and CD38-negative cells were separated by a wide margin. While attempting this procedure, its important to follow exact cell counting and timings of medium change.
Omitting medium change even one single day, or reducing the volume of that medium at any step, may cause massive cell death. Velocity of the rocking culture of embryoid bodies has to be carefully optimized.
