Kiyoko Izawa

Establishment of Novel Embryonic Stem Cell Lines Derived from the Common Marmoset (Callithrix jacchus)

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage‐specific embryonic antigen (SSEA)‐3, SSEA‐4, TRA‐1‐60, and TRA‐1‐81. On the other hand, SSEA‐1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34+ hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.

Antiangiogenic activity of BAI1 in vivo: implications for gene therapy of human glioblastomas.

Glioblastomas are the most common primary brain tumors in adults. These tumors exhibit a high degree of vascularization, and malignant progression from astrocytoma to glioblastoma is often accompanied by increased angiogenesis and the upregulation of vascular endothelial growth factor and its receptors. In this study, we investigated the in vivo antiangiogenic and antitumor effects of brain-specific angiogenesis inhibitor 1 (BAI1) using human glioblastoma cell lines. Glioblastoma cells were transduced with an adenoviral vector encoding BAI1 (AdBAI1), and Northern and Western blot analyses, respectively, demonstrated BAI1 mRNA and protein expression in the transduced tumor cells. Using an in vivo neovascularization assay, we found that angiogenesis surrounding AdBAI1-transduced glioblastoma cells transplanted into transparent skinfold chambers of SCID mice was significantly impaired compared to control treated cells. Additionally, in vivo inoculation with AdBAI1 of established subcutaneous or intracerebral transplanted tumors significantly impaired tumor growth and promoted increased mouse survival. Morphologically, the tumors exhibited signs of impaired angiogenesis, such as extensive necrosis and reduced intratumoral vascular density. Taken together, these data strongly indicate that BAI1 may be an excellent gene therapy candidate for the treatment of brain tumors, especially human glioblastomas.

Vaccine effect of granulocyte-macrophage colony- stimulating factor or CD80 gene-transduced murine hematopoietic tumor cells and their cooperative enhancement of antitumor immunity

To develop immunogene therapy targeting minimal residual hematopoietic tumor cells in patients, we transduced murine GM-CSF or CD80 gene into murine WEHI 3B myelomonocytic leukemia and EL-4 thymic lymphoma cells using retroviral vectors and evaluated their effects on inducing antitumor responses in syngeneic host mice. Subcutaneously injected GM-CSF- and CD80 gene-transduced WEHI 3B (GMCSF/WEHI/3.2 or CD80/WEHI/1.8, respectively) cells lost their original tumorigenicity in immunocompetent syngeneic mice. Results from tumor inoculation experiments using athymic nude mice suggested that the rejection of GMCSF/WEHI/3.2 in immunocompet- ent mice depended fully on T cells and that of CD80/WEHI/1.8 depended partly on T cells and partly on NK cells. In both WEHI 3B and EL-4 models, irradiated GM-CSF gene-transduced cells provided strong immunoprotection against wild-type cells, but irradiated CD80 gene- transduced cells did not. A remarkably high cooperative effect was obtained when irradiated GMCSF/EL-4 and CD80/EL-4 were inoculated together. These results suggested that the tumor vaccine effect is efficiently enhanced by GM-CSF gene transduction and CD80 gene transduction induces some protective antitumor immunity in co operation with GM-CSF gene transduction.

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus‐glycoprotein‐pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood‐cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl‐overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals.

PurposeThe application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells.MethodsInterleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage.ResultsThe Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells.ConclusionAlthough in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity.

Gaussia Luciferase for Bioluminescence Tumor Monitoring in Comparison with Firefly Luciferase

Gaussia luciferase (Gluc) is a secreted reporter, and its expression in living animals can be assessed by in vivo bioluminescence imaging (BLI) or blood assays. We characterized Gluc as an in vivo reporter in comparison with firefly luciferase (Fluc). Mice were inoculated subcutaneously with tumor cells expressing both Fluc and Gluc and underwent Flue BLI, Gluc BLI, blood assays of Glue activity, and caliper measurement. In Gluc BLI, the signal from the tumor peaked immediately and then decreased rapidly. In the longitudinal monitoring, all measures indicated an increase in tumor burden early after cell inoculation. However, the increase reached plateaus in Gluc BLI and Fluc BLI despite a continuous increase in the caliper measurement and Gluc blood assay. Significant correlations were found between the measures, and the correlation between the blood signal and caliper volume was especially high. Gluc allows tumor monitoring in mice and should be applicable to dual-reporter assessment in combination with Fluc. The Gluc blood assay appears to provide a reliable indicator of viable tumor burden, and the combination of a blood assay and in vivo BLI using Glue should be promising for quantifying and localizing the tumors.

Evaluation of gadoxetate disodium as a contrast agent for mouse liver imaging : comparison with gadobenate dimeglumine

We investigated the characteristics of gadoxetate disodium (Gd-EOB-DTPA) as a contrast agent for magnetic resonance imaging of the mouse liver. Mice were imaged sequentially under isoflurane anesthesia using a T1-weighted, three-dimensional fast low-angle shot (3D FLASH) sequence after an intravenous injection of Gd-EOB-DTPA or gadobenate dimeglumine (Gd-BOPTA), and the time course of the contrast effect was examined. The time course of the contrast effect of Gd-EOB-DTPA was also assessed after intravenous injection under pentobarbital anesthesia and after subcutaneous injection while awake or under isoflurane or pentobarbital anesthesia. Moreover, different doses of Gd-EOB-DTPA or Gd-BOPTA were injected subcutaneously into conscious mice, and the clarity of the liver border was evaluated visually. Intravenous injection under isoflurane anesthesia caused rapid contrast enhancement in the liver with both Gd-EOB-DTPA and Gd-BOPTA, and the contrast effect was 41% stronger with Gd-EOB-DTPA. Subcutaneous injection of Gd-EOB-DTPA caused delayed but favorable contrast enhancement in the liver. Washout of Gd-EOB-DTPA was faster in mice injected while awake than in those injected under anesthesia. After intravenous injection, washout was faster under pentobarbital anesthesia than under isoflurane anesthesia. The peak liver contrast was 11% and 18% stronger under pentobarbital anesthesia than under isoflurane anesthesia, after intravenous and subcutaneous injections, respectively. Subcutaneous injection of Gd-EOB-DTPA or Gd-BOPTA caused dose-dependent contrast effects in the liver. At a given dose, the contrast effect tended to be stronger and liver demarcation tended to be clearer with Gd-EOB-DTPA than with Gd-BOPTA. In conclusion, intravenous or subcutaneous injection of Gd-EOB-DTPA produces a favorable contrast effects in the mouse liver, indicating its potential in investigating mouse models of liver diseases. The contrast effects vary between conscious mice and anesthetized mice and among anesthetic agents used.

MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset.

Abstract. A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes.

In vivo leukemogenic potential of an interleukin 7 receptor α chain mutant in hematopoietic stem and progenitor cells

Somatic gain-of-function mutations in interleukin 7 receptor α chain (IL7Rα) have been described in pediatric T and B acute lymphoblastic leukemias (T/B-ALLs). Most of these mutations are in-frame insertions in the extracellular juxtamembrane-transmembrane region. By using a similar mutant, a heterozygous in-frame transmembrane insertional mutation (INS), we validated leukemogenic potential in murine hematopoietic stem/progenitor cells, using a syngeneic transplantation model. We found that ectopic expression of INS alone in hematopoietic stem/progenitor cells caused myeloproliferative disorders, whereas expression of INS in combination with a Notch1 mutant led to the development of much more aggressive T-ALL than with wild-type IL7Rα. Furthermore, forced expression of INS in common lymphoid progenitors led to the development of mature B-cell ALL/lymphoma. These results demonstrated that INS has significant in vivo leukemogenic activity and that the lineage of the resulting leukemia depends on the developmental stage in which INS occurs, and/or concurrent mutations.