RESUMEN
We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.
Asunto(s)
Neoplasias del Colon/terapia , Células Madre Embrionarias/citología , Antígenos de Histocompatibilidad/genética , Interferón beta/metabolismo , Células Mieloides/trasplante , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Células Madre Embrionarias/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Asesinas Naturales/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Mieloides/citología , Células Mieloides/metabolismo , Trasplante Homólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although first-line chemotherapy has a high rate of complete responses in ovarian cancer patients, the vast majority of patients present with recurrent disease that has become refractory to conventional chemotherapy. Peritoneal dissemination and malignant ascites are the hallmarks of recurrent or advanced ovarian cancer and severely reduce quality of life. Development of therapeutic measures to treat such patients is eagerly anticipated. Macrophage infiltration is observed in various types of cancer including epithelial ovarian cancer. In addition, macrophages are involved in the formation of spheroids in the malignant ascites of ovarian cancer and promote cancer growth. iPS-ML, macrophage-like myelomonocytic cells generated from human induced pluripotent stem (iPS) cells, made close contacts with ovarian cancer cells in vitro. We hypothesized that, if we inoculate iPS-ML-producing IFN-ß (iPS-ML/IFN-ß) into the peritoneal cavity of patients with ovarian cancer, IFN-ß produced by the iPS-ML/IFN-ß would efficiently act on the cancer cells to suppress cancer growth. To evaluate this hypothesis, we injected iPS-ML/IFN-ß into SCID mice bearing peritoneally disseminated human ovarian cancer cells, SKOV3. Immunohistochemical analysis of the intraperitoneal tumors detected iPS-ML/IFN-ß infiltrating into the cancer tissues. Therapy with iPS-ML/IFN-ß significantly suppressed tumor progression. In addition, dramatic reduction of cancer-related ascites was observed. Collectively, it is suggested that iPS-ML/IFN-ß therapy offers a new approach for the treatment of patients with advanced ovarian cancer.
Asunto(s)
Ascitis/terapia , Interferón beta/metabolismo , Monocitos/trasplante , Neoplasias Ováricas/terapia , Neoplasias Peritoneales/terapia , Animales , Ascitis/etiología , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/inmunología , Ratones , Ratones SCID , Monocitos/citología , Monocitos/inmunología , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/inmunología , Neoplasias Peritoneales/complicaciones , Neoplasias Peritoneales/inmunología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We established a method to generate a large quantity of myeloid lineage cells from mouse embryonic stem (ES) cells, termed ES cell-derived proliferating myeloid cell lines (ES-ML). ES-ML continuously proliferated in the presence of M-CSF and GM-CSF. ES-ML genetically modified to express an anti-HER2 (neu) mAb single-chain V region fragment reduced the number of cocultured mouse Colon-26 cancer cells expressing HER2. Stimulation of ES-ML with IFN-γ plus LPS or TNF resulted in almost complete killing of the Colon-26 cells by the ES-ML, and the cytotoxicity was mediated, in part, by NO produced by ES-ML. When ES-ML were injected into mice with i.p. established Colon-26 tumors, they efficiently infiltrated the tumor tissues. Injection of ES-ML with rIFN-γ and LPS inhibited cancer progression in the mouse peritoneal cavity. Coinjection of TNF-transfected or untransfected ES-ML with rIFN-γ inhibited cancer growth and resulted in prolonged survival of the treated mice. In this experiment, transporter associated with Ag processing (TAP)1-deficient ES-ML exhibited therapeutic activity in MHC-mismatched allogeneic recipient mice. Despite the proliferative capacity of ES-ML, malignancy never developed from the transferred ES-ML in the recipient mice. In summary, TAP-deficient ES-ML with anticancer properties exhibited a therapeutic effect in allogeneic recipients, suggesting the possible use of TAP-deficient human-induced pluripotent stem cell-derived proliferating myeloid cell lines in cancer therapy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Neoplasias del Colon/terapia , Células Madre Embrionarias/inmunología , Macrófagos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interferón gamma/farmacología , Lipopolisacáridos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Madre Pluripotentes/inmunología , Receptor ErbB-2/inmunología , Anticuerpos de Cadena Única/inmunología , Trasplante HomólogoRESUMEN
Adoptive cell transfer (ACT) is a type of personalized immunotherapy in which expanded immune cells are administered to patients with cancer. However, single-cell populations, such as killer T cells, dendritic cells, natural killer (NK) cells, and NKT (NKT) cells, have been generally used, and their effectiveness remains limited. Here, we established a novel culture method via CD3/CD161 co-stimulation and successfully expanded CD3+/CD4+ helper T cells, CD3+/CD8+ cytotoxic T cells (CTLs), CD3-/CD56+ NK cells, CD3+/CD1d+ NKT cells, CD3+/CD56+ NKT cells, CD3+/TCRγδ+ T cells, and CD3-/CD11c+/HLA-DR+ dendritic cells in peripheral blood mononuclear cells from healthy donors; their respective numbers were 155.5, 1132.5, 5.7, 117.0, 659.2, 325.6, and 6.8 times higher than those before expansion. These mixed immune cells showed strong cytotoxicity against cancer cell lines Capan-1 and SW480. Moreover, both CD3+/CD8+ CTLs and CD3+/CD56+ NKT cells killed tumor cells in cell contact-dependent and -independent manners via granzyme B and interferon-γ/TNF-α, respectively. Furthermore, the cytotoxicity of the mixed cells was significantly superior to that of CTLs or NKTs alone. A bet-hedging CTL-NKT circuitry is one potential mechanism underlying this cooperative cytotoxicity. Collectively, CD3/CD161 co-stimulation may be a promising culture method to expand multiple, distinct immune cell populations for the treatment of cancer.
Asunto(s)
Células T Asesinas Naturales , Neoplasias , Humanos , Complejo CD3 , Linfocitos T CD8-positivos , Células Asesinas Naturales , Neoplasias/terapia , Neoplasias/metabolismo , Linfocitos T Citotóxicos/metabolismo , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
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.
Asunto(s)
Diferenciación Celular , Células Dendríticas/citología , Macrófagos/citología , Células Madre Pluripotentes/citología , Animales , Presentación de Antígeno/inmunología , Línea Celular , Forma de la Célula , Reactividad Cruzada/inmunología , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Epítopos/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenotipo , Células Madre Pluripotentes/metabolismo , Propiedades de Superficie , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
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 beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-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-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m 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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Células Dendríticas/inmunología , Células Madre Embrionarias/inmunología , Epítopos de Linfocito T/inmunología , Antígenos H-2/genética , Activación de Linfocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/genética , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/prevención & control , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/prevención & control , Citotoxicidad Inmunológica/genética , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Femenino , Antígenos H-2/biosíntesis , Antígenos H-2/metabolismo , Haplotipos/inmunología , Prueba de Histocompatibilidad , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Unión Proteica/genética , Unión Proteica/inmunología , Linfocitos T Citotóxicos/metabolismo , Microglobulina beta-2/deficienciaRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Células Dendríticas/inmunología , Células Madre Embrionarias/inmunología , Animales , Diferenciación Celular/inmunología , Línea Celular , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Vectores Genéticos/inmunología , Vectores Genéticos/metabolismo , Humanos , Macaca fascicularis , Ratones , Transfección/métodosRESUMEN
BACKGROUND: iPS-ML are myeloid lineage cells with a proliferative capacity derived from induced pluripotent stem (iPS) cells. This study aimed to examine therapeutic effect of iPS-ML producing interferon-ß (iPS-ML/IFN-ß) towards primary and metastatic liver cancer and investigate the mechanism of that effect. METHODS: We established a xenograft model of liver metastasis by injecting the spleen of SCID mice with MKN-45 human gastric cancer cells and also a primary liver cancer model by injecting SK-HEP-1 human hepatocellular carcinoma cells into the liver. After cancer lesions were established, iPS-ML/IFN-ß was administered by intraperitoneal injection, and therapeutic effect was evaluated. RESULTS: The i.p. injection of iPS-ML/IFN-ß resulted in a significant retardation of cancer progression and prolonged mouse survival. The infiltration of i.p. administered iPS-ML into tumor lesions located below the liver capsule was observed, suggesting tumor-directed migration and penetration of the liver capsule by iPS-ML. The IFN-ß concentration in the liver was maintained at levels sufficient to exert an anti-cancer effect for at least 3 days post-injection, accounting for the potent therapeutic effect obtained by injection two to three times per week. CONCLUSIONS: This study demonstrates the therapeutic potential of the iPS-ML/IFN-ß in patients with liver cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/terapia , Células Madre Pluripotentes Inducidas/trasplante , Interferón beta/uso terapéutico , Neoplasias Hepáticas/terapia , Células Mieloides/trasplante , Neoplasias Gástricas/terapia , Animales , Carcinoma Hepatocelular/secundario , Tratamiento Basado en Trasplante de Células y Tejidos , Modelos Animales de Enfermedad , Neoplasias Hepáticas/secundario , Ratones SCID , Neoplasias Gástricas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Interferón Tipo I/fisiología , Melanoma/terapia , Células Mieloides/metabolismo , Neoplasias Cutáneas/terapia , Animales , Diferenciación Celular , Línea Celular Tumoral , Humanos , Inmunoterapia , Macrófagos/inmunología , Melanoma/inmunología , Melanoma/secundario , Ratones SCID , Trasplante de Neoplasias , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4+ and CD8+ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8+ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.
Asunto(s)
Antígenos/biosíntesis , Células Dendríticas/citología , Receptores de Lipopolisacáridos/inmunología , Adulto , Diferenciación Celular , Proliferación Celular , Células Dendríticas/inmunología , Femenino , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.
Asunto(s)
Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular , Células Mieloides/citología , Células Mieloides/inmunología , Células Madre Pluripotentes/citología , Traslado Adoptivo , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/metabolismo , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunofenotipificación , Melanoma/inmunología , Melanoma/patología , Melanoma/terapia , Ratones , Células Mieloides/metabolismo , Neoplasias/inmunología , Péptidos/inmunología , Fenotipo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer's 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 Aß added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aß. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aß. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aß-degrading activity. In vitro, expression of NEP2 but not anti-Aß scFv enhanced the effect to reduce the level of soluble Aß oligomer in the culture medium and to alleviate the neurotoxicity of Aß. 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 Aß 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.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Células Madre Pluripotentes Inducidas/citología , Macrófagos/metabolismo , Neprilisina/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Macrófagos/citología , Macrófagos/inmunología , Ratones , Microscopía Fluorescente , Neprilisina/genética , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Autoinmunidad , Células Dendríticas/trasplante , Diabetes Mellitus Tipo 1/terapia , Células Madre Embrionarias/citología , Encefalomielitis Autoinmune Experimental/terapia , Células TH1/inmunología , Animales , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
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 ß significantly inhibited the growth of human gastric and pancreatic cancers implanted in the peritoneal cavity of immunocompromised mice.
RESUMEN
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-ß (iPS-ML/IFN-ß) significantly inhibited the intra-peritoneal growth of NUGC-4 cancer. Furthermore, iPS-ML/IFN-ß 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.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Interferón beta/metabolismo , Células Mieloides/metabolismo , Células Mieloides/trasplante , Neoplasias Peritoneales/terapia , Trasplante de Células Madre , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Proliferación Celular , Citocinas/farmacología , Humanos , Inyecciones Intraperitoneales , Macrófagos/patología , Ratones , Ratones SCID , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Neoplasias Peritoneales/secundario , Peritoneo/metabolismo , Peritoneo/patología , Receptor ErbB-2/metabolismo , Anticuerpos de Cadena Única/metabolismoRESUMEN
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.