Notch signaling is required for generation of conventional type 1 dendritic cells from human induced pluripotent stem cells

Abstract

Abstract Compelling evidence shows a critical role of conventional type 1 dendritic cells (cDC1) in T-cell mediated antitumor immunity; however, obtaining large numbers of cDC1 is difficult. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous cDC1. However, generation of cDC1 from human iPSC remains elusive. Here, we hypothesized that Notch signaling would facilitate generation of cDC1 from human iPSC. We found that human iPSC gave rise to CD141+XCR1+CLEC9A+HLA-DR+ cells on OP9 feeder cells expressing the Notch ligand delta-like 1 (OP9-DL1) while the majority of iPSC-derived cells differentiated on OP9 cells were monocytic cells. Single-cell RNA sequencing analyses confirmed the presence of cDC1 as well as identified the heterogeneity of iPSC-derived CD1c+ cells; cDC2A, cDC2B, CD5+cDC2, and DC3. Inhibition of Notch signaling during co-culture of iPSC-derived CD34+ hematopoietic progenitor cells with OP9-DL1 cells abrogated generation of cDC1, and made expression of CD5, CLEC4A, CLEC10A and CD163 in CD1c+ DC similar to those differentiated on OP9 cells. Notch-activated human iPSC-derived XCR1+CLEC9A+HLA-DR+CD11c+ cells exhibited similar gene expression profile with peripheral blood cDC1, and phagocytotic, T-cell proliferative, and cytokine producing functions. Our study demonstrates that Notch signaling is required for the differentiation of cDC1-like cells, and facilitates generation of various cDC subsets from human iPSC. These findings provide insights into the future development of personalized treatment with unlimited numbers of autologous cDC1 from human iPSC. This work was supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park’s Flow and Image Cytometry, Bioinformatics, and Genomic Shared Resources. This work was supported by the Melanoma Research Alliance and the Sarcoma Foundation of America (F. Ito), Uehara Memorial Foundation (T. Oba), and National Cancer Institute (NCI) grant, U24CA232979 (M. Long and S. Liu), K08CA197966 and R01CA255240-01A1 (F. Ito).