iPSC-Derived Monocytes Generate Functional M1 and M2 Macrophages with Enhanced Cytokine Secretion and Tumor Cell-Killing Activity

Ian Hay1, Christopher B. Rohde1, Matthew Angel1

1Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 30, No 4S1, 2022

Cancer immunotherapy has advanced rapidly over the past two decades, with several autologous chimeric antigen receptor (CAR)-T cell therapies approved for the treatment of hematologic cancers. However, CAR-T cells have shown limited activity against solid tumors, in part due to the immunosuppressive nature of the tumor microenvironment preventing CAR-T cell infiltration. This has led to investigation of other immune cells as alternatives to T-cell-based therapies, including monocytes and monocyte-derived macrophages, which exhibit innate tumor-infiltration properties. We developed a process for differentiating pluripotent stem cells along a myeloid lineage, and generated populations of cells with characteristics of monocytes and M1 and M2 macrophages, including cytokine secretion and tumor cell-killing activity. mRNA-reprogrammed human induced pluripotent stem cells (iPSCs) were differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells were harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1×104 cells per cm2 per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. iPSC-derived monocytes showed similar expression of CD11b, CD14, CD33, CD45, and CD163 compared to PBMC-derived monocytes, and increased expression of markers indicative of an activated state: CD80 and CD206. Compared to PBMC-derived monocytes, iPSC-derived monocytes showed both higher viability in culture and superior recovery from cryopreservation. iPSC-derived monocytes were further differentiated into macrophages by exposure to MCSF for 3-4 days, and were assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages were polarized with interferon gamma (IFN-γ, 50 ng/mL) and lipopolysaccharide (LPS, 10 ng/mL) for 48 hours, while M2 macrophages were treated with IL-4 (10 ng/mL) for 48 hours. iPSC-derived monocytes differentiated into macrophages with >90% efficacy, as assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). M1 and M2 polarized iPSC-derived macrophages secreted similar levels of TNFα, IL-12p70, and IL-10 compared to PBMC-derived macrophages. iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro after 24 hours at an E:T ratio of 5:1. We demonstrate a process for differentiating mRNA-reprogrammed iPSCs into cytotoxic macrophages. The mRNA reprogramming and differentiation processes are virus-free and DNA-free, avoiding any potential risk of vector integration. These results suggest that mRNA-reprogrammed iPSCs may represent a viable source of macrophages for the development of therapies to treat various indications, including solid tumors.