Yukoh Nakazaki

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.

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.

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.

TARC and RANTES enhance antitumor immunity induced by the GM-CSF-transduced tumor vaccine in a mouse tumor model

IntroductionTransduction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene into mouse tumor cells abrogates their tumorigenicity in vivo. Our previous report demonstrated that gene transduction of GM-CSF with either TARC or RANTES chemokines suppressed in vivo tumor formation. In this paper, we examined whether the addition of either recombinant TARC or RANTES proteins to irradiated GM-CSF-transduced tumor vaccine cells enhanced antitumor immunity against established mouse tumor models to examine its future clinical application.Materials and methodsThree million irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated into syngeneic WEHI3B-preestablished BALB/c mice.ResultsVaccinations were well tolerated. Mice treated with GM-CSF-transduced cells and the chemokines demonstrated significantly longer survival than mice treated with GM-CSF-transduced cells alone. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-γ, and TNF-α, suggesting enhanced innate and adaptive immunity. Immunohistochemical analysis of tumor sections after vaccination revealed a more significant contribution of CD4+ and CD8+ T cells to tumor repression in the combined vaccine groups than controls.ConclusionsTARC and RANTES enhance the immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and may be clinically useful.

GM-CSF and B7-1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumor immunity in syngeneic mice

B7‐1 (CD80) co‐stimulatory molecule gene–transduced Lewis lung carcinoma (LLC) cells (LLC/B7 cells) resulted in remarkable loss of tumorigenicity in syngeneic C57BL/6 mice (87.5% rejection) compared to B7‐negative, wild‐type LLC (LLC/wt) cells (0% rejection). However, mice that had rejected LLC/B7 cells developed almost no systemic immunity protective against challenge with wild‐type tumor cells after 4 weeks (11.8% rejection). Enhancement of MHC class I (H‐2Kb) expression of LLC/B7 cells with in vitro interferon‐γ treatment did not result in enhancement of protective immunity. In vivo depletion assay revealed that abrogation of tumorigenicity in LLC/B7 depended on CD8+ T cells but not on CD4+ T cells. However, vaccination of C57BL/6 mice with irradiated LLC cells transduced with GM‐CSF (LLC/GM) led to the induction of potent, specific immunity against challenge with the LLC/wt cells after 2 weeks (80.8% rejection). Next, we established a double transfectant of LLC cells expressing both B7‐1 and GM‐CSF (LLC/GM + B7). The tumorigenicity of these clonal cells was also remarkably suppressed (90% rejection) to the same degree as LLC/B7, whereas that of LLC/GM was not suppressed (0% rejection). Interestingly, mice that had rejected LLC/GM+B7 cells developed enhanced protective immunity against challenge with LLC/wt cells after 4 weeks (55.6% rejection) compared to the results of LLC/B7 cells (11.8%). To evaluate whether co‐expression of GM‐CSF and B7‐1 enabled the tumor cells to activate cytotoxic T cells more efficiently than B7‐1 alone, we performed an in vitro killing assay. We found that immunization with LLC/GM+B7 cells resulted in a 3‐fold stronger cytotoxic response than that with LLC/B7. Our data indicate that co‐transfection of the B7‐1 co‐stimulatory molecule and GM‐CSF genes may be more effective for the induction of stronger protective immunity in this experimental system. Int. J. Cancer 73:556–561, 1997.

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate.

p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53‐responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti‐cancer therapy.

Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells

Abstract There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears promising. Before initiating a clinical study using an ex vivo gene-transduced autologous cell vaccine-based immunogene therapy for RCC in Japan, in 1992 we initially planned a Japanese version of a clinical protocol in collaboration with a US group. In 1993, the original protocol was refined. We performed five preclinical qualification studies using RCC nephrectomy specimens from patients in 1997, and the results showed that preparation of RCC cells for autologous vaccines at the Clinical Cell Technology Facility, Research Hospital of the Institute of Medical Science, University of Tokyo, was feasible. Subsequently in August 1998, the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sport approved our clinical protocol. We have recruited two patients with stage IV RCC to our study so far. Here we report the background to the initiation of cancer gene therapy in Japan.

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.

Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors

Abstract: To establish a new non‐human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti‐human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid‐specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c‐kit mAbs could be used to purify the marmoset haematopoietic progenitor cells.

TCR-Vβ repertoire analysis with RT-PCR was useful for the early detection of pulmonary relapsed T-cell lymphoma after autologous peripheral blood stem cell transplantation

Pulmonary recurrence of malignant lymphoma is a rare event after stem cell transplantation. We report here a 45‐year‐old male who was successfully diagnosed with relapsed pulmonary T‐cell lymphoma using an RT‐PCR method. Clonal expansion of T cells expressing identical TCR V‐D‐J junction size (Vβ5‐Jβ1.5) was demonstrated in lymphocyte groups obtained from both bronchoalveolar lavage fluid at relapse, and paraffin embedded lymph node samples resected when he was first diagnosed with angioimmunoblastic T‐cell lymphoma. This method provided evidence to diagnose relapsed pulmonary angioimmunoblastic T‐cell lymphoma in its early phase. Am. J. Hematol. 64:124–127, 2000.