Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

The overall goal of this procedure is to generate and characterize T lymphocytes from induced pluripotent stem or IPS cells. The IPS cells can then be differentiated in vitro on an OP nine DL one culture system and then matured in vivo in a rag deficient mouse. Or the cells can be genetically engineered to over express OT one TCR and then adoptively transferred for in vivo development.

After six weeks of in vivo differentiation, the mice can be challenged by intraperitoneal injection with EEG seven tumor cells and then the antigen specificity of the IPS derived T cells can be evaluated. Ultimately, the IPS cells can differentiate into both conventional and antigen specific T cells, the latter of which are able to protect animals from tumor challenge. The implications of this technique extend toward individualized the cancer immunotherapy.

The method allows a generation of large numbers of tumor reactive T cells derives from minimum amount of patient's blood or skin tissue On day zero transfer five times 10 to the fourth IPS cells onto a 100 millimeter culture dish containing a confluent OP nine, DL one cell monolayer in 20%FBS alpha MEM media on day five, trypsin eyes. Then centrifuge the cells after incubating them on a fresh 100 millimeter culture dish for 30 minutes and 37 degree incubator plate, five times 10 to the fifth of the floating cells in 20%FBS alpha, MEM media and mouse flat three ligand on a new 100 millimeter dish containing a monolayer of OP nine. DL one cells on day eight gently pipette down the loosely attached cells, centrifuge the cells, and then transfer the cells to a single well of a six well culture plate coated with confluent OP nine DL one cells incubate the cells at 37 degrees Celsius, 5%carbon dioxide for two more weeks on day 22.

Incubate detached IPS cells in fresh media on a new culture dish at 37 degrees Celsius for 30 minutes. Then remove about half of the floating cells and pass them through a 70 micron nylon strainer to exclude cell clumps and wash the resulting single cell suspension three times with cold PBS. After the third wash, resuspend the cells in PBS at 1.5 times 10th of the seventh cells per milliliter and maintain the cells on ice before IV injection.

Place a four week old rag deficient mouse under an infrared light to dilate the tail vein. Then fill a one milliliter syringe equipped with a 26 and a half gauge needle with 200 microliters of the cell suspension and adoptively transfer the cells through the dilated tail vein. Three weeks later after confirming euthanization of the adoptively transferred mouse, remove the lymph nodes and spleen after mechanically breaking down the tissues.

Lyce the single cell suspension with a CK lysis buffer and then wash the resulting mononucleo cytes twice in cold PBS. Then after staining for cell surface markers, evaluate the cells by flow cytometric analysis on day zero, use the gene JAMA transfection reagent to transfect overnight plated plate E packaging cells with an OT one MIDR on day one seed one times 10 to the six IPS cells into one well of a 0.1%gelatin pre-coded 24 well plate on day two, collect the pseudo virus containing S natin from the plat E cells and then pass it through a 0.4 micron filter to exclude potential contaminants after centrifuge. Transducing the cells in the presence of poly brain for one hour at 330 times G at 32 degrees Celsius.

Incubate them overnight at the same temperature after repeating the transduction procedure. Trypsin eyes the transduced IPS cells on day four and then after pelleting, the cells resus suspend the cells in fresh media and seed them on pre-coded irradiated snl 7 6 7 feeder cells. Once the transduced cells have reached con fluency, sort the GFP and DS red double positive cells by flow cytometry.

Six weeks after the adoptive transfer of the IPS cells inject 50 microliters of EEG seven thoma cells into the peritoneal cavity of the same mouse on day 50 of the tumor challenge. Use a mil E biotech CD eight positive T cell isolation kit to sort CD eight positive T cells from the spleen and lymph nodes of the adoptively transferred animal. Mix the isolated CD eight positive T cells with irradiated SPOC size from a naive C 57 black six J mouse at a one to 10 ratio and pulse the co-culture with 0.5 micromoles per mil of ova for 40 hours.

Thereafter, add felden A to the cells for another four hours. Finally, evaluate the cell populations by flow cytometric analysis After isolating SP cytes from a naive C 57 black six J mouse. Split the cell suspension into two groups.

Label the CFSE high group with five micromoles per mil of CFSE and pulse the cells with 10 micrograms per mil of ova peptide for one hour, labeled A-C-F-S-E low group with 0.5 MICROMOLES per mil CFSE and do not pulse. Mix 2.5 times 10 to the sixth, the CFSE high cells with 2.5 times 10 to the sixth, the CFSE low cells in PBS. Then analyze the cytes by flow cytometry for CFSE expression.

16 hours after adoptive transfer into the mouse of interest To count intraperitoneal tumor cells on day 20 of the tumor challenge. Use a 10 milliliter syringe equipped with an 18 and a half gauge needle and cold PBS to lavage the peritoneal cavity. Count the recovered tumor cells for identifying tumor infiltrating cells.

Remove the tumor at a late stage of the tumor challenge and then cut the tumor into three pieces. Place the first tumor piece into a cryo vial and place the vial on dry ice. Immediately fix the second piece formaldehyde.

Preserve the third piece in conditioned RPMI 1640 media after air drying the cryopreserved tissue sections. Fix them in cold acetone for 15 minutes after air drying the fixed sections for another 15 minutes. Wash the slides for five minutes in PBS after the wash.

Place the slides in a moist chamber and cover the tissue sections with 30 microliters of 3%B, SA and PBS for 30 minutes to block non-specific binding. Then blot off the blocking buffer and incubate the tissue sections with a 50 microliter mixture of pe anti TCR RV alpha two antibody and fitzy anti ova antibody diluted in 3%PSA in PBS in the moist chamber for two hours at the end of incubation. Wash the slides three times in cold PBS finally mount the sections with a water-based mounting media before fluorescent microscopic examination for flow cytometric analysis of tumor infiltrating T cells.

Squash the tumor into a single cell suspension. Then analyze the cells for specific surface markers. C CD three and TCR beta are used as markers of T cells to determine whether stimulation of IPS cells with the notch ligand DL one could contribute to T cell CD four and CD eight cell surface expression on CD three positive TCR r beta positive IPS cell derived cells was assessed.

Note the dot plot on the right which shows day 22 CD three positive TCR R beta positive CD four negative CD eight positive single positive T cells generated from IPS cells in vitro. In addition, the IPS cell derived single positive cells from the previous figure produced interleukin two and interferon gamma when stimulated in vitro by plate coated anti CD three and soluble anti CD 28 antibodies confirming that the IPS cell derived T cells are functional after adoptive transfer into recipient mice. The majority of TCR gene transduced IPS cells underwent differentiation into CD eight positive CTLs, which responded in vitro to peptide stimulation by secreting interleukin two and interferon gamma in these dot plots from pooled lymph node and spleen cells.

CD eight positive V beta five positive T cells were generated in mice adoptively transferred with TCR Transduced but not control transduced IPS cells. The CD eight positive V beta five positive populations were positive for intracellular cytokine staining for interleukin two and interferon gamma production represented in these histograms by the dark lines as compared to the shaded isotype control histograms indicating that the IPS derived CTLs were functional. These data show that CFSE high cells pulsed with eptide represented by the peaks on the right in each graph and CFSE low control cells represented by the peaks on the left that were injected into mice 10 weeks after IPS cell transfer or one day after O OT one CTL transfer.

The antigen specific IPS derived CTLs responded to antigen stimulation and had cytotoxic activity as indicated by the absence of CFSE high cells here. OT one TCR gene transduced IPS cells were adoptively transferred into C 57 black six mice and then the mice were subjected to challenge with EEG seven tumor cells on day 20 tumor cells in the peritoneal cavity were enumerated. Note the significant reduction in tumor cell numbers in mice that received TCR TRANSDUCED IPS compared to the control groups.

Most importantly, adoptive transfer of TCR TRANSDUCED IPS cells triggered infiltration of overreactive CTLs into tumor tissues and protected animals from tumor challenge as seen in this h and e staining of tumor tissues recovered days 30 to 35 after tumor challenge tumors from mice adoptively transferred with TCR transduced cells or OT one CTLs but not mock injected or control. Transduced cells were infiltrated by inflammatory cells as indicated by the red arrows here. Ova specific V alpha two positive CTLs in red were found to infiltrate ova expressing tumor tissues in green.

While tumors in mice from the control groups had fewer or no tumor infiltrating cells in this figure, single cell suspensions from tumor tissues were analyzed for expression of V alpha two positivity and V beta five positivity by flow cytometry after gating on the CD eight positive population. Note that tumor tissues from mice adoptively transferred with TCR Transduced IPS cells exhibited the highest level of tumor infiltrating cells while tumors from mice that received control. Transduced IPS exhibited no infiltration by ova specific CD eight positive T cells.

After watching this video, you should have a good understanding of how to generate antigen specific T cells from IPS cells and how to evaluate the functions of the IPS derived the T cells.