RESUMEN

The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.

Asunto(s)

Transformación Celular Neoplásica/metabolismo , Células Madre Embrionarias/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Teratoma/metabolismo , Secuencia de Aminoácidos , Animales , Transformación Celular Neoplásica/patología , Células Cultivadas , Células Madre Embrionarias/patología , Humanos , Macaca fascicularis , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Especificidad de Órganos , Especificidad de la Especie , Teratoma/patología , Trasplante Heterólogo

RESUMEN

OBJECTIVE: Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS: From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS: In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION: Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Animales , Linaje de la Célula , Células Cultivadas/trasplante , Genes Reporteros , Vectores Genéticos/análisis , Supervivencia de Injerto , Macaca fascicularis , Osteoblastos/citología , Células del Estroma/trasplante , Acondicionamiento Pretrasplante , Trasplante Autólogo

RESUMEN

Sendai virus (SeV) vectors can introduce foreign genes efficiently and stably into primate embryonic stem (ES) cells. For the application of these cells, the control of transgene expression is important. Cynomolgus ES cells transduced with a SeV vector expressing the green fluorescent protein (GFP) gene were propagated in Knockout serum replacement (KSR)-supplemented medium, used widely for the serum-free culture of ES cells, and growth and transgene expression were evaluated. The SeV vector-mediated GFP expression was suppressed in the KSR-supplemented medium, although it was stable in regular fetal bovine serum (FBS)-supplemented medium. Propagation in the KSR-supplemented medium eventually resulted in a complete suppression of GFP expression and eradication of the SeV genome. The inhibitory effect of KSR on the transduction was attributable to the positive selection of untransduced ES cells in addition to the removal of the SeV vector from transduced cells. KSR also reduced the efficiency of the transduction. SeV vector-mediated transgene expression in ES cells was suppressed in the KSR-supplemented medium. Although the suppression is limited in specified cells such as ES cells, these findings will help elucidate how to control transgene expression.

Asunto(s)

Medios de Cultivo/química , Células Madre Embrionarias/fisiología , Vectores Genéticos , Macaca fascicularis , Virus Sendai/metabolismo , Transducción Genética , Animales , Bovinos , Células Cultivadas , Células Madre Embrionarias/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos BALB C , Virus Sendai/genética , Transgenes

RESUMEN

Because embryonic stem (ES) cells are able to proliferate indefinitely and differentiate into any type of cell, they have the potential for providing an inexhaustible supply of transplantable cells or tissues. However, methods for the in vitro differentiation of human ES cells are still quite limited. One possible strategy would be to generate differentiated cells in vivo. In view of future clinical application, we investigated the possibility of using xenogeneic large animals for this purpose. We transplanted nonhuman primate cynomolgus ES cells into fetal sheep at 43-67 gestational days (full term 147 days, n=15). After birth, cynomolgus tissues, which were mature teratomas, had been engrafted in sheep when more than 1 x 10(6) ES cells were transplanted at <50 gestational days. Despite the sustained engraftment, both cellular and humoral immune responses against the ES cells were detected, and additional transplantation was not successful in the animals. At 2 weeks post-transplantation, the ES cell progeny proliferated when transplanted at 48 (<50) gestational days, whereas they were cleared away when transplanted at 60 (>50) gestational days. These results support the rapid development of the xenogeneic immunological barrier in fetal sheep after 50 gestational days. Notably, a large number of Foxp3(+) regulatory T cells were present around the ES cell progeny, but macrophages were absent when the transplant was conducted at <50 gestational days, implying that regulatory T cells and premature innate immunity might have contributed to the sustained engraftment. In conclusion, long-term macroscopic engraftment of primate ES cells in sheep is feasible despite the xenogeneic immunological barrier.

Asunto(s)

Transferencia de Embrión , Células Madre Embrionarias/trasplante , Supervivencia de Injerto , Macaca fascicularis , Ovinos , Útero , Adaptación Biológica/genética , Adaptación Biológica/inmunología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Células Cultivadas , Transferencia de Embrión/métodos , Embrión de Mamíferos , Desarrollo Embrionario/genética , Células Madre Embrionarias/fisiología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Datos de Secuencia Molecular , Embarazo , Ovinos/embriología , Quimera por Trasplante , Inmunología del Trasplante , Trasplante Heterólogo , Útero/fisiología

RESUMEN

Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/?cnull (NOG) mice, teratomas developed in all the animals transplanted with 1 × 105 or more cells, but were not observed in any mouse transplanted with 1 × 103 cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 × 105. When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 × 105. When cynomolgus ES cell-derived progenitor cells (1 × 106), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.

RESUMEN

Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/gammac(null) (NOG) mice, teratomas developed in all the animals transplanted with 1 x 10(5) or more cells, but were not observed in any mouse transplanted with 1 x 10(5) cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 x 10(5). When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 x 10(5). When cynomolgus ES cell-derived progenitor cells (1 x 10(6)), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.

Asunto(s)

Células Madre Embrionarias/trasplante , Trasplante de Células Madre/efectos adversos , Teratoma/etiología , Trasplante Heterólogo/efectos adversos , Animales , Células Madre Embrionarias/patología , Inyecciones Intramusculares , Inyecciones Subcutáneas , Macaca fascicularis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Músculo Esquelético , Teratoma/patología

RESUMEN

Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP>KL, and KKK>AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP>KL, and KKK>AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP>KL, and KKK>AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.

Asunto(s)

Transporte Activo de Núcleo Celular , ADN Complementario/metabolismo , ADN Viral/metabolismo , Integrasa de VIH/fisiología , Animales , Células COS

RESUMEN

Z-100 is an arabinomannan extracted from Mycobacterium tuberculosis that has various immunomodulatory activities, such as the induction of interleukin 12, interferon gamma (IFN-gamma) and beta-chemokines. The effects of Z-100 on human immunodeficiency virus type 1 (HIV-1) replication in human monocyte-derived macrophages (MDMs) are investigated in this paper. In MDMs, Z-100 markedly suppressed the replication of not only macrophage-tropic (M-tropic) HIV-1 strain (HIV-1JR-CSF), but also HIV-1 pseudotypes that possessed amphotropic Moloney murine leukemia virus or vesicular stomatitis virus G envelopes. Z-100 was found to inhibit HIV-1 expression, even when added 24 h after infection. In addition, it substantially inhibited the expression of the pNL43lucDeltaenv vector (in which the env gene is defective and the nef gene is replaced with the firefly luciferase gene) when this vector was transfected directly into MDMs. These findings suggest that Z-100 inhibits virus replication, mainly at HIV-1 transcription. However, Z-100 also downregulated expression of the cell surface receptors CD4 and CCR5 in MDMs, suggesting some inhibitory effect on HIV-1 entry. Further experiments revealed that Z-100 induced IFN-beta production in these cells, resulting in induction of the 16-kDa CCAAT/enhancer binding protein (C/EBP) beta transcription factor that represses HIV-1 long terminal repeat transcription. These effects were alleviated by SB 203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), indicating that the p38 MAPK signalling pathway was involved in Z-100-induced repression of HIV-1 replication in MDMs. These findings suggest that Z-100 might be a useful immunomodulator for control of HIV-1 infection.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , VIH-1/efectos de los fármacos , Lípidos/farmacología , Macrófagos/efectos de los fármacos , Mananos/farmacología , Antígenos CD4/biosíntesis , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Eliminación de Gen , Productos del Gen env/deficiencia , Genes nef/genética , Vectores Genéticos , VIH-1/genética , VIH-1/fisiología , Humanos , Interferón beta/biosíntesis , Macrófagos/virología , Mycobacterium tuberculosis/química , Receptores CCR5/biosíntesis , Transcripción Genética/efectos de los fármacos , Transfección , Replicación Viral/efectos de los fármacos

RESUMEN

We investigated the effects of signaling through CD28 family molecules on human immunodeficiency virus type 1 (HIV-1) replication in vitro. A monoclonal antibody (mAb) specific for inducible costimulator (ICOS) suppressed both X4 and R5 HIV-1 replication in CD4(+) peripheral blood mononuclear cells (PBMC). This suppression was not attributable to reduced cell growth or viability. CD28 mAb showed variable effects and also suppressed HIV-1 replication when immobilized. Replication of pseudotype viruses with HIV-1-but not with vesicular stomatitis virus G-envelope was efficiently suppressed in CD4(+) PBMC treated with ICOS or CD28 mAbs. However, CD4, CXCR4, and CCR5 expression on the surface was not down-regulated. Moreover, HIV-1 replication in CD4(+) PBMC was suppressed by a soluble form of human B7-H2, a ligand of ICOS, but was enhanced by soluble B7-1, a ligand for CD28. These findings suggest that natural or artificial ligands for ICOS potentially suppress HIV-1 replication mainly at the entry stages.

Asunto(s)

Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antivirales/farmacología , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/patología , División Celular , Supervivencia Celular , Citocinas/biosíntesis , VIH-1/efectos de los fármacos , Humanos , Técnicas In Vitro , Proteína Coestimuladora de Linfocitos T Inducibles , Isoxazoles/farmacología , Leflunamida , Ligandos , Ratones , Receptores del VIH/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunología

RESUMEN

RNA interference (RNAi) is a sequence-specific RNA degradation process mediated by short double-stranded RNAs termed small interfering RNAs. Here, we describe the lentivirus-based vector small interfering RNA system expressing. As a pilot study, we generated constructs expressing small hairpin RNA (shRNA) specific for luciferase gene (shLuc) or green fluorescence protein (shGFP) under the control of human H1 RNA polymerase III promoter. The effect of the shRNA was evaluated against HIV-1 infection in a single-round or multiple-round infectious system using an HIV-1 molecular clone carrying the luc or GFP gene. In the single-round infectious system, cells transduced with shLuc by lentiviral vector significantly reduced (approximately 90% reduction) viral gene expression after challenge infection at a multiplicity of infection of 10. These transduced cells continued to resist against at least four sequentially repeated challenge infections. Importantly, this efficient antiviral activity persisted over 35 days in culture. In a multiple-round infectious system using a replication-competent HIV-1 molecular clone carrying the GFP gene, we also observed that a lentiviral vector expressing shGFP could inhibit HIV-1 replication for at least 1 week. The profound effect of lentiviral shRNA was also observed in human primary monocyte-derived macrophages. Thus, shRNA introduced through the lentiviral vector can be useful for efficient and stable gene suppression in human cells including primary non-dividing cells. Moreover, quantitative analysis of viral cDNA synthesis on challenge infection showed that viral genomic RNAs packaged in incoming virus core might not be targeted by shLuc. Instead, the degradation of transcripts from integrated proviral DNAs might be a major cause of the profound reduction in HIV-1 gene expression by shRNA in our system.

Asunto(s)

Silenciador del Gen , Vectores Genéticos , VIH-1/genética , Lentivirus/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismo , División Celular , Línea Celular , Línea Celular Transformada , Regulación Viral de la Expresión Génica , Humanos , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , Recombinación Genética , Replicación Viral