Abstract
For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now “classic” OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.
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Acknowledgments
The authors thank Génève Awong and Paul Oleynik for expertise in cell sorting; Jenny Suh Yoon Park, Dylan Lee, and Anita Tia for technical support; and Elaine Herer and Rose Kung from the Women & Babies Program at Sunnybrook Health Sciences Centre (Toronto, ON, Canada) for their ongoing support by providing umbilical cord blood samples. This work was supported by grants from the Canadian Institutes of Health Research (CIHR FDN-154332), the Canadian Cancer Society Research Institute (CCSRI-705960), and the Krembil Foundation. J.C.Z.-P. was supported by a Canada Research Chair in Developmental Immunology.
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Mohtashami, M., Brauer, P.M., Zúñiga-Pflücker, J.C. (2023). Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines. In: Bosselut, R., Vacchio, M.S. (eds) T-Cell Development. Methods in Molecular Biology, vol 2580. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2740-2_15
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DOI: https://doi.org/10.1007/978-1-0716-2740-2_15
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