A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs

Fong, H.; Mendel, M.; Jascur, J.; Najmi, L.; Kim, K.; Lew, G.; Garimalla, S.; Schock, S.; Hu, J.; Villegas, A.; Conway, A.; Fontenot, J.; Zompi, S.

doi:10.1101/2023.07.01.547333

bioRxiv · 10.1101/2023.07.01.547333

A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs

Fong, H.·Mendel, M.·Jascur, J.·Najmi, L.·Kim, K.·Lew, G.·Garimalla, S.·Schock, S.·Hu, J.·Villegas, A.·Conway, A.·Fontenot, J.·Zompi, S.

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Abstract

iPSCs can serve as a renewable source of a consistent edited cell product, overcoming limitations of primary cells. While feeder-free generation of clinical grade iPSC-derived CD8 T cells has been achieved, differentiation of iPSC-derived CD4sp and regulatory T cells requires mouse stromal cells in an artificial thymic organoid. Here we report a serum- and feeder-free differentiation process suitable for large-scale production. Using an optimized concentration of PMA/Ionomycin, we generated iPSC-CD4sp T cells at high efficiency and converted them to Tregs using TGF{beta} and ATRA. Using genetic engineering, we demonstrated high, non-viral, targeted integration of an HLA-A2 CAR in iPSCs. iPSC-Tregs +/- HLA-A2-targeted CAR phenotypically, transcriptionally and functionally resemble primary Tregs and suppress T cell proliferation in vitro. Our work is the first to demonstrate an iPSC-based platform amenable to manufacturing CD4 T cells to complement iPSC-CD8 oncology products and functional iPSC-Tregs to deliver Treg cell therapies at scale.

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Fong, H., Mendel, M., Jascur, J., Najmi, L., Kim, K., Lew, G., Garimalla, S., Schock, S., Hu, J., Villegas, A., Conway, A., Fontenot, J., Zompi, S.. 2023-07-02. A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs. https://doi.org/10.1101/2023.07.01.547333

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