Feeder-free differentiation and expansion for T cells from induced pluripotent stem cells

Clinical ecacy demonstrated by chimeric antigen receptor T cell therapy call for further development that could broaden their applicability. One such direction is to develop alternate T-cell sources and T cells differentiated from pluripotent stem cells may be an ideal candidate. The present protocol provides a feeder-free and scalable method to generate T lymphocytes from induced pluripotent stem cells. we developed feeder-free differentiation


Introduction
Recent clinical e cacy obtained with chimeric antigen receptor T cell therapy indicated that T cells could be used to treat a wide range of diseases. The development of "off-the-shelf" T cells will further expand their applications. T cells derived from induced pluripotent stem cells (iPSCs) may became an ideal cell source for this purpose 1 . Although our group and others have demonstrated therapeutic e cacy of iPSCderived T cells, they are differentiated on murine stromal cell lines and are proliferated on irradiated human peripheral blood mononuclear cells, raising safety concerns and scalable issues [2][3][4][5][6][7] . To overcome these issues, we have developed a feeder-free differentiation protocol to generate and proliferate T cells from induced pluripotent stem cells. • α-MEM basal medium Prepare according to the manufacturer's instruction. Divide into 210-ml aliquots and store at 4 °C for up to 2 weeks.
• FACS buffer FACS buffer contains 2% (vol/vol) FBS in D-PBS. Store at 4 °C for up to 6 months.
• MTG solution Add 87 μl of MTG to 10 ml of endotoxin-free reagent-grade distilled water. Mix well and divide into 500-μl aliquots. Store for up to 6 months at -20°C.
• Ascorbic acid solution Add 50 mg of ascorbic acid to 1 ml of endotoxin-free reagent-grade distilled water. Dissolve completely, divide into 100-μl aliquots and store for up to 6 months at -20°C.
• Reconstitution of cytokines Reconstitute cytokines according to the product information provided by manufacturer.

Day 4:
Harvest the differentiating EBs as described in day 1 and resuspend them in 2 ml EB basal medium supplemented with 50 ng/ml rhVEGF, 50 ng/ml rhbFGF and 50 ng/ml rhSCF per well.
From days 9-14: Harvest the differentiating cultures and replace the spent medium with fresh day 7 medium for every 2-3 days.
Day 38: Collect the cells and resuspend them in maturation medium without OKT3 and incubate for 4 days.

T-cell proliferation
Day 41: On the day before T-cell activation, coat 48-well plates with CD3/Retronectin solution composed of 3.0 µg/ml anti-human CD3 and 150 µg/ml Retronectin at 4 °C overnight.

Troubleshooting Time Taken
Anticipated Results