One of the most challenging aspects of studying PDAC, or pancreatic cancer, is the availability of an experimental model that can recapitulate the progression of PDAC from the early PanIN stages to the aggressive state. The other challenge is to experimentally capture plasticity and heterogeneity of PDAC. So using PDAC iPS cells will help us tackle some of these challenges and expand our experimental tools to study this disease.
We have established a robust protocol to reprogram PDACs to iPS cells. By generating more iPS lines from many more PDAC patients, we can study the transition of pancreatic cancer from early to advanced stages, and see the commonalities and differences between these lines. This will help us discover early diagnostic markers that cover more patients and generate drugs that target the different states of the disease.
The interesting question that came out of reprogramming PDAC to iPS cells is what makes this cancer so resistant to cell fate conversion compared to normal cells. Is this due to specific genes or genetic mutations that tell us something about cancer cell identity? To begin, see the HEK293T cells 24 hours before transfection in a 15-centimeter dish containing GMEM.
Incubate the cells at 37 degrees Celsius and 5%carbon dioxide until they reach 40 to 50%confluency. Label three 15-milliliter plastic tubes with the appropriate lentivirus name. Add 1.710 milliliters of reduced serum medium and 90 microliters of transfection reagent to each tube.
Mix the contents by vortexing before incubating the tubes at room temperature. After five minutes, add the packaging vector mixture to the transfection medium and vortex. Then, add 7.5 micrograms of reprogramming vector and the pWPT-GFP control to the respective transfection mixture.
Vortex the tubes for two seconds before incubating for 15 minutes at room temperature. To transfect the 293T cells with each lentivirus, dropwise add a transfection DNA mixture into the dish. Incubate the transfected cells at 37 degrees Celsius and 5%carbon dioxide.
After 14 to 16 hours, replace the medium with 30 milliliters of fresh 293T medium and continue incubation for 60 to 72 hours. Observe the cells daily and check transfection efficiency. Next, transfer the virus transfection culture into 15-milliliter tubes and spin down at 1, 932 G for 10 minutes at 4 degrees Celsius.
Filter the lentivirus supernatant using a 0.45-micron syringe filter into a new 50-milliliter tube. One day before lentivirus transduction, prepare two wells of a six-well plate containing 100, 000 PDAC cells per well. Prepare two 15-milliliter tubes with 2 milliliters of prewarmed PDAC medium.
In the first tube, add 12 milliliters of reprogramming lentivirus. Then, add 12 microliters of polybrene and mix by pipetting. To the second tube, add 2 microliters of polybrene.
Now carefully remove the medium from each well of a six-well plate and wash once with PBS at room temperature. Add the reprogramming infection medium from tube one to the first well. Add the mixture from tube two to the second well.
Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen. The next day, replace the medium from both wells with fresh PDAC medium and continue incubation for up to 48 hours as demonstrated. To begin, coat the six-well plate with 2 milliliters of 0.2%gelatin solution per well.
Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. Next, dropwise add IMEFs into a 15-milliliter tube containing 10 milliliters of pre-warmed human fiberblast medium and mix by gently inverting the tube. Centrifuge the cell suspension at 309 G for three minutes, and remove the supernatant before resuspending the pellet in 12 milliliters of human fibroblast medium.
Place 2 milliliters of the cell suspension into each well of the gelatin-coated six-well plate, and incubate overnight at 37 degrees Celsius and 5%carbon dioxide. Discard the medium from the lentivirus-infected and non-infected PDAC and wash the cells twice with PBS. Then, add 500 microliters of trypsin to dissociate the cells and incubate at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen for 15 minutes.
Centrifuge the cell suspension at 300 G for five minutes and resuspend the pellet in 1 milliliter of PDAC medium. Wash the IMEF plate with PDAC medium. Then, add 50, 000 lentivirus-infected PDAC cells per well in five wells of IMEF plate and incubate overnight as demonstrated previously.
After preparing the reprogramming medium, add 100 microliters of basic fibroblast growth factor to 100 milliliters of reprogramming medium. The next day, wash the cells growing on IMEF feeders twice with prewarmed reprogramming media. Then, add 2 milliliters of media to each well and incubate overnight at 37 degrees Celsius with 5%carbon dioxide and 3%oxygen.
After passing the iPSC pool five times, observe the robust colonies maintaining ESC-like morphology. The PDAC, BxPC-3, H6c7, and human fibroblast, show different days of reprogramming to form a mature ESC-like morphology. Clonal iPSC lines were established from each reprogramming of PDAC, BxPc3, H6c7, and human fibroblast cells.
All established iPSC lines stained positive for TRA-160, confirming their reprogramming to pluripotency.
