Miwa Haruta

Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells.

Methods have been established to generate dendritic cells (DCs) from mouse and human embryonic stem (ES) cells. We designated them as ES‐DCs and mouse models have demonstrated the induction of anti‐cancer immunity and prevention of autoimmune disease by in vivo administration of genetically engineered ES‐DCs. For the future clinical application of ES‐DCs, the histoincompatibility between patients to be treated and available human ES cells and the ethical concerns associated with human ES cells may be serious obstacles. However, recently developed induced pluripotent stem (iPS) cell technology is expected to resolve these issues. This report describes the generation and characterization of DCs derived from mouse iPS cells. The iPS cell‐derived DCs (iPS‐DCs) possessed the characteristics of DCs including the capacity of T‐cell‐stimulation, antigen‐processing and presentation and cytokine production. DNA microarray analyses revealed the upregulation of genes related to antigen‐presenting functions during differentiation into iPS‐DCs and similarity in gene expression profile in iPS‐DCs and bone marrow cell‐derived DCs. Genetically modified iPS‐DCs expressing antigenic protein primed T‐cells specific to the antigen in vivo and elicited efficient antigen‐specific anti‐tumor immunity. In addition, macrophages were generated from iPS cells (iPS‐MP). iPS‐MP were comparable with bone marrow cell‐derived macrophages in the cell surface phenotype, functions, and gene expression profiles. Stem Cells 2009;27:1021–1031

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid β (Aβ), the causal protein of Alzheimer’s disease. The scFv-transfectant iPS-MP exhibited efficient Aβ-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Genetically Manipulated Human Embryonic Stem Cell‐Derived Dendritic Cells with Immune Regulatory Function

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen‐specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell‐derived DC (ES‐DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES‐DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES‐DC. ES‐DC introduced with invariant chain‐based antigen‐presenting vectors by this procedure stimulated HLA‐DR‐restricted antigen‐specific T cells in the absence of exogenous antigen. Forced expression of programmed death‐1‐ligand‐1 in ES‐DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES‐DC. Generation and genetic modification of ES‐DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES‐DC technology is therefore considered to be a novel means for immune therapy.

TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells

We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b+ myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells ‘iPS-ML’, and the iPS-ML-derived APC ‘ML-DC’. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8+ T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8+ T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

Activation of Antigen-Specific Cytotoxic T Lymphocytes by β2-Microglobulin or TAP1 Gene Disruption and the Introduction of Recipient-Matched MHC Class I Gene in Allogeneic Embryonic Stem Cell-Derived Dendritic Cells

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or β2-microglobulin (β2m) gene was disrupted in 129 (H-2b)-derived ES cells and then expression vectors for the H-2Kd or β2m-linked form of Kd (β2m-Kd) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1−/−/Kd ES-DC and β2m−/−/β2m-Kd ES-DC. As intended, both of the transfectant ES-DC expressed Kd but not the intrinsic H-2b haplotype-derived MHC class I. β2m−/−/β2m-Kd and TAP1−/−/Kd ES-DC were not recognized by pre-activated H-2b-reactive CTL and did not prime H-2b reactive CTL in vitro or in vivo. β2m−/−/β2m-Kd ES-DC and TAP1−/−/Kd ES-DC had a survival advantage in comparison to β2m+/−/β2m-Kd ES-DC and TAP1+/+/Kd ES-DC, when transferred into BALB/c mice. Kd-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2b haplotype-encoded MHC class I. β2m−/−/β2m-Kd ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than β2m+/−/β2m-Kd ES-DC. The modification of the β2m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

Therapeutic Effect of Human iPS-Cell-Derived Myeloid Cells Expressing IFN-β against Peritoneally Disseminated Cancer in Xenograft Models

We recently developed a method to generate myeloid cells with proliferation capacity from human iPS cells. iPS-ML (iPS-cell–derived myeloid/macrophage line), generated by introducing proliferation and anti-senescence factors into iPS-cell–derived myeloid cells, grew continuously in an M-CSF–dependent manner. A large number of cells exhibiting macrophage-like properties can be readily obtained by using this technology. In the current study, we evaluated the possible application of iPS-ML in anti-cancer therapy. We established a model of peritoneally disseminated gastric cancer by intraperitoneally injecting NUGC-4 human gastric cancer cells into SCID mice. When iPS-ML were injected intraperitoneally into the mice with pre-established peritoneal NUGC-4 tumors, iPS-ML massively accumulated and infiltrated into the tumor tissues. iPS-ML expressing IFN-β (iPS-ML/IFN-β) significantly inhibited the intra-peritoneal growth of NUGC-4 cancer. Furthermore, iPS-ML/IFN-β also inhibited the growth of human pancreatic cancer MIAPaCa-2 in a similar model. iPS-ML are therefore a promising treatment agent for peritoneally disseminated cancers, for which no standard treatment is currently available.

Degradation of amyloid beta by human induced pluripotent stem cell-derived macrophages expressing Neprilysin-2

The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimers disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.

Suppression of Th1-Mediated Autoimmunity by Embryonic Stem Cell-Derived Dendritic Cells

We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.

Application of iPS cell-derived macrophages to cancer therapy.

We established a method to produce a large quantity of myeloid cells from human inducible pluripotent stem cells (iPSCs). When injected intraperitoneally into mice carrying established peritoneal tumors, iPSC-derived myeloid cells (iPS-MCs) efficiently accumulated within neoplastic lesions. The intraperitoneal injection of iPS-MCs expressing interferon β significantly inhibited the growth of human gastric and pancreatic cancers implanted in the peritoneal cavity of immunocompromised mice.

Generation of a large number of functional dendritic cells from human monocytes expanded by forced expression of cMYC plus BMI1.

Anticancer vaccination therapies with monocyte-derived dendritic cells (DC) are widely conducted. A large number of primary monocytes (approximately 10(8) cells) are needed to generate the number of DC required to achieve an effect upon vaccination, and monocytes are usually purified from peripheral blood mononuclear cells obtained by apheresis procedure, which is somehow invasive for cancer patients. As a means to facilitate the generation of DC for therapeutic use, we herein report a method to amplify human monocytes. We found that lentivirus-mediated transduction of cMYC along with BMI1 induced proliferation of CD14(+) monocytes derived from 9 out of 12 blood donors, and we named the monocyte-derived proliferating cells CD14-ML. Their proliferation continued for 3-5 weeks in the presence of M-CSF and GM-CSF, resulting in 20-1000-fold amplification. Importantly, the expanded CD14-ML differentiated into fully functional DC (CD14-ML-DC) upon the addition of IL-4 to the culture. We successfully stimulated autologous CD8(+) T cells with CD14-ML-DC pulsed with cytomegalovirus peptide or MART-1 peptide to generate antigen-specific CTL lines. This is the first report describing the method for in vitro expansion of human peripheral blood monocytes.