Induced pluripotent stem cells reprogrammed from primary dendritic cells provide an abundant source of immunostimulatory dendritic cells for use in immunotherapy

Abstract

Cell types differentiated from induced pluripotent stem cells (iPSCs) are frequently arrested in their development program, more closely resembling a fetal rather than an adult phenotype, potentially limiting their utility for downstream clinical applications. The fetal phenotype of iPSC-derived dendritic cells (ipDCs) is evidenced by their low expression of MHC class II and costimulatory molecules, impaired secretion of IL-12, and poor responsiveness to conventional maturation stimuli, undermining their use for applications such as immune-oncology. Given that iPSCs display an epigenetic memory of the cell type from which they were originally derived, we investigated the feasibility of reprogramming adult DCs to pluripotency to determine the impact on the phenotype and function of ipDCs differentiated from them. Using murine bone marrow-derived DCs (bmDCs) as proof of principle, we show here that immature DCs are tractable candidates for reprogramming using non-integrating Sendai virus for the delivery of Oct4, Sox2, Klf4, and c-Myc transcription factors. Reprogramming efficiency of DCs was lower than mouse embryonic fibroblasts (MEFs) and highly dependent on their maturation status. Although control iPSCs derived from conventional MEFs yielded DCs that displayed a predictable fetal phenotype and impaired immunostimulatory capacity in vitro and in vivo, DCs differentiated from DC-derived iPSCs exhibited a surface phenotype, immunostimulatory capacity, and responsiveness to maturation stimuli indistinguishable from the source DCs, a phenotype that was retained for 15 passages of the parent iPSCs. Our results suggest that the epigenetic memory of iPSCs may be productively exploited for the generation of potently immunogenic DCs for immunotherapeutic applications.

Keywords: dendritic cell; epigenetic memory; immunostimulation; immunotherapy; induced pluripotent stem cell; maturation.

Figure 1

iPSC_MEF‐dendritic cells (DCs) display a primitive, fetal phenotype. A, Phase‐contrast photomicrograph of iPSC_MEF‐DCs developing in clusters around the perimeter of embryoid bodies (EBs). Inset: High magnification (×40) of individual iPSC‐derived dendritic cells (ipDCs). B, Flow cytometric analysis of iPSC_MEF‐DCs, showing expression of CD11c, CD80, CD86, and MHC class II (red histograms). Blue histograms represent background staining with isotype‐matched control monoclonal antibodies (mAbs). C, IL‐10 secretion by bone marrow‐derived DCs (bmDCs) and iPSC_MEF‐DCs in response to challenge with lipopolysaccharide (LPS). D, Secretion of IL‐12 by bmDCs and iPSC_MEF‐DCs in response to challenge with 1 μg/mL LPS. C, D, Red bars denote bmDCs, blue bars represent iPSC_MEF‐DCs. All data are representative of multiple experiments and are reported as mean values with error bars representing SD. **P < .001

Figure 2

Characterization of source bone marrow‐derived dendritic cells (bmDCs) prior to reprogramming. A, CD11c expression of cells (red histograms) harvested at day 7 from cultures of bone marrow prior to and following the purification of DCs using CD11c‐labeled magnetic beads. Blue histograms represent background staining with an isotype control mAb. B, Phase‐contrast photomicrograph of bmDCs following purification showing typical veiled and dendritic morphology. C, Flow cytometric analysis of CD11c‐purified bmDCs showing expression of CD40, CD54, CD86, and MHC class II (red histograms). Blue histograms represent background staining with appropriate isotype controlled mAbs. D, IL‐12 secretion by CD11c‐purified bmDCs in response to increasing concentrations of lipopolysaccharide (LPS). All data reported as mean values with error bars representing SD of replicates. E, Immunostimulatory capacity of source bmDCs as a function of the proliferation of allogeneic T cells assessed by progressive dilution of CFSE. The unfilled histogram represents cocultures of DCs and allogeneic T cells whereas the blue histogram denotes unstimulated control T cells. All data are representative of multiple independent experiments

Figure 3

Reprogramming of bone marrow‐derived dendritic cells (bmDCs) to pluripotency. A, Phase‐contrast photomicrograph of a typical colony of induced pluripotent stem cells (iPSCs) reprogrammed from bmDCs (iPSC_DC) at passage 7, cultured on mouse embryonic fibroblast (MEF) feeder cells. B, Phase‐contrast photomicrograph of a typical colony of iPSCs reprogrammed from MEFs (iPSC_MEF), cultured on feeder cells, likewise at passage 7. C‐F, Methylene blue staining of six well plates revealing the abundance of iPSC colonies from a representative experiment in which equivalent numbers of MEFs (C) and bmDCs (D and F) were reprogrammed in parallel. E, Control well of MEFs without the addition of reprogramming vectors. F, Well containing equivalent numbers of bmDCs induced to mature in response to lipopolysaccharide (LPS) prior to reprogramming. Numbers in the bottom left hand corner of each image denote the number of iPSC colonies observed

Figure 4

Characterization of iPSC_DC demonstrating that full pluripotency can be achieved from source bone marrow‐derived dendritic cells (bmDCs). A, Phase‐contrast photomicrograph of a typical iPSC_DC colony cultured on gelatin‐coated flasks demonstrating characteristic nuclear morphology and the prominent nucleoli, typical of pluripotent cells. B, Photomicrograph of EBs at day 10 of culture, derived from iPSC_DC at passage 6. Approximately 34% of EBs were found to contract spontaneously in suspension culture (see Supplementary Online Video S1). C, Flow cytometric analysis of iPSC_DC demonstrating expression of the pluripotency markers SSEA‐1, Oct4, and Nanog (red histograms). Blue histograms represent isotype‐matched control mAbs. D, Phase‐contrast photomicrographs of tissues differentiated in vitro from iPSC_DC, showing tissues derived from each of the three embryonic germ layers: mesoderm (cardiomyocytes: the broken line denotes the region of spontaneously contracting tissue), endoderm (gut epithelium), and ectoderm (optic cup identified by the preponderance of retinal pigmented epithelial cells, arrows). E, Hematoxylin and eosin (H&E) stained histological sections of teratomas formed following the engraftment of EBs derived from iPSC_DC under the kidney capsule of syngeneic mice. Photomicrographs show examples of tissues representing the three embryonic germ layers: Ectoderm (retinal pigment epithelium, cornified epithelium [arrows]), mesoderm (smooth muscle, bone), and endoderm (gut epithelium, goblet cells [arrow])

Figure 5

Characterization of iPSC‐derived dendritic cells (ipDCs) differentiated from iPSC_DC. A, Phase contrast photomicrographs of ipDCs differentiated from iPSC_DC prior to maturation (left hand image) and following maturation in response to lipopolysaccharide (LPS) (right hand image). B, Flow cytometric analysis of early passage iPSC_DC‐DC, demonstrating a baseline expression of MHC class II, CD40, CD54, CD80, and CD86 (red histograms), and their upregulation following coculture with 1 μg/mL LPS. Blue histograms represent background staining with appropriate isotype‐matched control mAbs. C, Comparison of IL‐12 secretion by bone marrow‐derived dendritic cells (bmDCs) and iPSC_DC‐DCs at passage 5 in response to challenge with LPS. Background levels of IL‐12 in the absence of LPS are shown as a broken line. D, Comparison of IL‐10 secretion by bmDCs and iPSC_DC‐DCs passage 5 in response to LPS. The dashed line denotes the background levels of IL‐10 detected in the absence of LPS. E, Processing and presentation of HEL by iPSC_DC‐DCs measured as a function of IL‐2 secretion by the 1C5.1 T‐cell hybridoma, specific for HEL_46‐61/H‐2A^k. All data are representative of multiple experiments and represent mean values with error bars denoting SD

Figure 6

iPSC‐derived dendritic cells (ipDCs) differentiated from iPSC_DC display an adult phenotype compared to their iPSC_MEF‐derived counterparts. A, Proportion of EBs derived from iPSC_DC and iPSC_MEF (both at passage 7) capable of generating ipDCs over a 16 day culture period. B, Flow cytometric analysis of CD40, CD86, and MHC class II expression (red histograms) by iPSC_MEF‐DCs, iPSC_DC‐DCs, and control bone marrow‐derived dendritic cells (bmDCs either immature or following maturation in response to lipopolysaccharide (LPS). Yellow histograms represent staining of immature bmDCs; blue histograms denote background staining with isotype‐matched control mAbs. C, Comparison of the immunostimulatory capacity of ipDCs differentiated from iPSC_DC and iPSC_MEF measured as a function of the proliferation of CFSE‐labeled allogeneic T cells (unfilled histograms). Blue histograms represent control T cells cultured in the absence of DCs. D, in vivo stimulation of CD4⁺ T cells following the i.v. administration of iPSC_DC‐DCs, iPSC_MEF‐DCs, or phosphate‐buffered saline (PBS) alone to female A1.RAG1^−/− mice. T cell activation is shown as a function of the upregulation of CD69, an early activation marker 20 hours after administration. All data are representative of multiple independent experiments