从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

The overall goal of this experiment is to generate induced pluripotent stem cells or iPSCs from melanoma infiltrating lymphocytes. This method can help answer key questions in the field of tumor immunology especially for cancer immunotherapy. The main advantage of this technique is that the ability to generate human iPS cells from tumor-infiltrating lymphocytes could provide unlimited numbers of less differentiated tumor specific T cells for immune therapy.

This technique may have applications for the treatment of metastatic melanoma as currently the poor survival of T cells in a clinic has been the major limitation of adaptive T cell therapy. This method may also be useful in the investigation of the T cell receptor repertoire in the tumor microenvironment. Demonstrating the procedure will be Dr.Kumiko Iwabuchi, a post doctoral fellow from my laboratory.

After obtaining the tumor material select five to 20 grams of tumor specimens and use scissors to dissect the normal, solid, and firm tissue from the fragile and or bloody necrotic areas of the tumor samples. Mince the dissected specimens as finely as possible then use a dissociator and a human tumor dissociation kit to disperse the minced tumor pieces into a single cell suspension according to the manufacturer's instructions. At the end of the dissociation filter the cells through 70 micron strainer into a 50 milliliter tube and wash the strainer two times with two milliliters of RPMI 1640.

Then pellet the cells by centrifugation and re-suspend the cells in 10 milliliters of fresh T-cell medium. To separate the cells by density gradient first layer 30 milliliters of 75%gradient solution followed by 10 milliliters of 100%gradient solution in DPBS without calcium or magnesium into a new 50 milliliter tube. Then carefully overlay the cells onto the gradient solution taking care not to disrupt the layers and centrifuge the sample.

At the end of separation, transfer the enriched tumor-infiltrating lymphocytes or TILs at the interface between the gradient solutions into a new 50 milliliter conical tube and wash the cells with 20 to 30 milliliters of fresh DPBS. Re-suspend the pellet in 10 milliliters of fresh T-cell medium, then add two milliliters of TILs per well to five wells of a six well plate and supplement the TIL cultures with Recombinant Human IL-2 for five days at 37 degrees Celsius and 5%carbon dioxide. On day five, change half the medium in each well and every two to three days thereafter.

When the cultures reach 80 to 90%confluency use a pipette to gently re-suspend the cells in each well and split the cultures at a one-to-two ratio in one milliliter of fresh T-cell medium supplemented with Recombinant Human IL-2. To prepare mitomycin-C-treated SNL feeder plates, first culture SNL feeder cells in 10 milliliters of SNL feeder cell medium on a 0.1%gelatin coated 10 centimeter dish. When the cells are 80 to 90%confluent, treat the feeders with mitomycin C for two hours and 15 minutes at 37 degrees Celsius in 5%carbon dioxide.

At the end of incubation, wash the cells two times with five milliliters of DPBS. Then detach the feeders with room-temperature Trypsin-EDTA. After one minute neutralize the dissociation reaction with 4.5 milliliters of fresh SNL feeder medium and transfer the single cell suspension into a 15 milliliter conical tube for centrifugation.

Re-suspend the pellet in 10 milliliters of fresh SNL feeder cell medium for counting. Then plate 1.5 times 10 to the six SNL feeder cells per well in 10 centimeter dishes and incubate the cultures at 37 degrees Celcius in 5%carbon dioxide for up to three days. To generate the iPSCs with Sendai Virus transfer five times 10 to the fifth TILs per well to a new six well plate coated with mouse anti-human CD3 in two milliliters of fresh T cell medium supplemented with Recombinant Human IL-2 and soluble mouse anti-human CD28.

Activate the TILs in the cell culture incubator for five days. Then use a pipette to pool the cultures in a 15 milliliter conical tube for counting. Next transfer one times 10 to the fifth TILs per well into a 24 well plate coated with anti-human CD3 in 500 microliters of reprogramming medium supplemented with soluble mouse anti-human CD28 in Recombinant Human IL-2.

After 24 hours in the cell culture incubator enumerate the cells in each well and prepare several volumes of virus at the appropriate multiplicities of infection. Then add a mixture of Sendai Virus vectors at 10 to 20 MOI into the appropriate volume of medium and add the virus containing medium to each well of TILs. For determination of the transduction efficiency add the calculated volume of Sendai Virus GFP into a well of activated TILs and return the cultures to the incubator for 24 hours.

The next day, view the Sendai Virus GFP infected cells by fluorescence microscopy to estimate the infection efficiency of the transduction. Pool the virally infected cells from each well into a 15 milliliter tube for centrifugation and re-suspend the pellet in 0.5 milliliters of human embryonic stem cell medium supplemented with primate embryonic stem cell medium and basic fibroblast growth factor. Next add 9.5 milliliters of fresh human embryonic stem cell medium to the cells and transfer the suspension onto a mitomycin C treated SNL feeder cell plate.

In this image TILs on day 21 of culture with Recombinant Human IL-2 that are ready to be activated with anti CD3 and CD28 are shown. The TILs can be transfected with Sendai Virus at a multiplicity of infection of 20. Here a typical pluripotent clone on SNL feeder cells 18 to 21 days after Sendai Virus infection is shown.

As observed in this representative karyogram the TIL derived iPSCs exhibit a normal karyotype. Immunofluorescence staining to confirm pluripotency marker expression on TIL-derived iPSCs demonstrates that these cells exhibit the expected levels of expression of typical pluripotency markers. In addition, iPSCs derived from melanoma TILs are able to form teratomas that contain a variety of cells from the three germ layers.

Further, TIL derived iPSCs retained their T cell receptor rearrangements as assessed by capillary electrophoresis. Our first development, this technique pave the way for researchers in the field of oncology to explore more effective cancer immunotherapies for metastatic melanoma patients. While attempting this procedure, it is important to check T cell proliferation and viability before Sendai Virus infection and to evaluate infection efficiency by the contour GFP expression.

After watching this video you should have a good understanding of how to generate human TIL cells from melanoma TILs.