The overall goal of this experiment is to develop a new method for generating stem cell-derived inflamed joint associated regulatory T cells for immunotherapy against autoimmune arthritis. This method can help answer key question in the autoimmunity field about potential immunotherpeutics for autoimmune arthritis. The main advantage of this technique is that the stem cell-derived regulatory T cells are nyped and of a single type.
Begin by diluting the feeder cells of interest to a minimum density of one times 10 the the fourth cells per square centimeter in OP9 medium, and plating one times 10 to the sixth feeder cells per 10 centimeter dish. When the cells become 80 to 90%confluent, remove the medium from the cultures and seed 0.5 to one times 10 to the fifth, retrovirally transduced inducible pluripotent stem cells or IPSCs in OP9 medium in each dish. Five days later, aspirate the medium and wash the cells with 10 milliliters of PBS.
Then, detach the cells with four milliliters of Trypsin per dish, at 37 degrees Celsius. After 10 minutes, stop the reactions with eight milliliters of IPSC medium and pull the cells for centrifugation. Resuspend the pellet in 10 milliliters of fresh IPSC medium and replate the cells onto new 10 centimeter dishes.
Allow the feeder cells to adhere to the plate for 30 minutes in the cell culture incubator. Then, collect the floating IPSCs and filter the transduced cells through a 70 micron strainer for counting. Seed five times 10 to the fifth IPSCs onto a fresh 80 to 90%confluent plate of feeder cells in OP9 medium supplemented with Murine-Flt3-Ligand and return the cultures to the incubator.
After three days, use a 10 milliliter pipette to wash the dish with the culture medium. Then, add five milliliters of fresh OP9 medium to the dish and gently wash off any semi-adherent IPSCs with careful, forceful pipetting that does not disrupt the feeder cell layer. Repeat the wash with 10 milliliters of PBS to harvest the last of the semi-adherent cells, pulling the washes from each dish into one conical tube per culture.
After spinning down the cells, resuspend the pellets in 10 milliliters of OP9 medium supplemented with Murine-Flt3-Ligand and IL-7 and filter the cells through a 70 micron strainer. Seed one IPSC culture per well into a six-well culture plate containing 80 to 90%confluent feeder cell monolayers and return the co-cultures to the incubator. Every two days, replace half of the medium with fresh OP9 medium supplemented with Murine-Flt3-Ligand and IL-7, and every four to six days, reseed the differentiating IPSCs onto new plates with a fresh layer of feeder cells as necessary.
Begin by detaching the cell cultures of interest with Trypsin and resuspending each cell type in 10 milliliters of fresh medium. Plate each cell culture in a new 10 centimeter dish and allow the feeder cells to adhere in the cell culture incubator. After 30 minutes, collect the floating IPSCs and pass the cultures through individual 70 micron cell strainers to remove the cell clusters for counting.
Resuspend each culture at a 1.5 times 10 to the seventh cells per milliliter concentration in cold PBS, filtering again as necessary. Then, place four to six-week-old female C57BL/6 mice one at a time in a small animal restrainer and adoptively transfer 200 microliters of one type of cell per mouse into the tail vein of each animal. Use a dial gauge caliper to measure the swelling of both knees to establish the baseline measurement.
10 days after the cell transfer, use a one milliliter syringe to inject the mice with 100 micrograms of methylated BSA emulsified in complete Freund's adjuvant at the base of the tail of each experimental animal. 17 days after the cell transfer, induce arthritis by intra-articular injection of 20 micrograms of methylated BSA in 10 microliters of PBS into the left knee joint and 20 micrograms of methylated BSA and 100 micrograms of whole Ovalbumin in 10 microliters of PBS into the right knee joint of each anesthetized adoptively transferred animal. Seven days after inducing the arthritis, measure the knees again for calculation of the percent increase in the knee diameter, and surgically excise the patellas.
Fix the joints in formalin. Then, after decalcification in EDTA, embed the knees in paraffin and obtain four micron sections for histochemical staining and analysis. On day 28, the antigen-specific T regulatory cells express substantial levels of CD3 and antigen-specific T cell receptors.
Most of these cells also express CD25, CD127, and CTLA-4, all of which are typically expressed at elevated levels in naturally occurring T regulatory cells. Indeed, FoxP3 persists in IPSC-derived T regulatory cells, even after long term in vitro stimulation with the Notch ligand, as detected by intracellular staining. In addition, these antigen-specific IPSC T regulatory cells produce suppressive cytokines when stimulated with antigen pulsed splenocytes in vitro, indicating a potential suppressive phenotype for these cells.
FoxP3 positive cells are observed in OVA treated knees with no FoxP3 positive cells apparent in knees that receive control vector transduced IPSCs. Further, many more CD4 positive, FoxP3 positive, TCR3 Beta 5 positive cells are visualized in the knees of mice that receive IPSCs transduced with the TCR-FoxP3 vector, than with the FoxP3 vector alone, suggesting that antigen-specific IPSC-Treg cells migrate to the antigen-induced arthritis knee after their adoptive transfer into recipient animals. IPSC-derived cell adoptive transfer also affects a substantial decrease in the inflammatory knee swelling when OVA is present, but has no effect on control methylated BSA injected knees.
The positive effects of the swelling reduction are further borne out by high resolution Micro-CT imaging of the reduced bone loss observed in cell transfer treated knees compared to control MBSA-only injected animals. Once mustered, these in-depth procedure can become completed in six weeks if it is performed properly. While attempting this technique, it is important to remember to induce the imbued differentiation of the TCR-FoxP3 antigen transduced IPSCs.
Following this procedure, different compounds of suppressive cytokines can also be deliberated to answer additional questions about the survival and quality of the antigen-specific IPSCs T reg cells. After it's development, this technique paved the way for researchers in the field of cell-based therapy to explore the role antigen-specific regulatory T cells in autoimmune disease. Don't forget that working with direct autobody bactors can be extremely hazardous and that precaution, such as working in a basic two-facility, should always be taken while performing a procedure.
Thanks for watching and good luck with your experiment.
