Yutaka Hanazono

Repair of Infarcted Myocardium Mediated by Transplanted Bone Marrow–Derived CD34+ Stem Cells in a Nonhuman Primate Model

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34+ cells were transplanted to the peri‐ischemic zone. To track the in vivo fate of transplanted cells, CD34+ cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline‐treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell–derived, GFP‐positive cells were found incorporated into the vascular structure, and GFP‐positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34+ cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell–derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.

Generation of naive-like porcine-induced pluripotent stem cells capable of contributing to embryonic and fetal development.

In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanakas 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Intramuscular injection of AAV-GDNF results in sustained expression of transgenic GDNF, and its delivery to spinal motoneurons by retrograde transport

Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not beta-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment.

Improved safety of hematopoietic transplantation with monkey embryonic stem cells in the allogeneic setting

Cynomolgus monkey embryonic stem cell (cyESC)‐derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC‐derived hematopoietic colony‐forming cells were detected in the newborns (4.1%–4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno‐transplanted with the same cyESC derivatives. It turned out that the cyESC‐derived donor cells included a residual undifferentiated fraction positive for stage‐specific embryonic antigen (SSEA)‐4 (38.2% ± 10.3%) despite the rigorous differentiation culture. When an SSEA‐4‐negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC‐derived hematopoietic engraftment was unperturbed (2.3%–5.0%). SSEA‐4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs.

Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells

Background. To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. Methods. Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. Results. A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (∼1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. Conclusions. Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they “leak” into an improper space such as the thoracic cavity.

Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.

Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice

The application of adeno-associated virus (AAV) vectors to cancers is limited by their low transduction efficiency. Previously, we reported that γ-ray enhanced the second-strand synthesis, leading to the improvement of the transgene expression, and cytocidal effect of the herpes simplex virus type-1 thymidine kinase (HSVtk) and ganciclovir (GCV) system. In this study, we extended this in vitro findings to in vivo. First, the laryngeal cancer cell line (HEp-2) and HeLa were treated with AAVtk/GCV, the number of surviving cells was reduced as the concentration of GCV increased. Furthermore, the 4u2009Gy irradiation enhanced the killing effects of AAVtk/GCV by four-fold on HeLa cells and 15-fold on HEp-2 cells. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AAV vectors in combination with γ-ray in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and significantly increased by γ-ray. The AAVtk/GCV system suppressed the tumors growth, and γ-ray augmented the antitumor activity by five-fold. These findings suggest that the combination of AAVtk/GCV system with radiotherapy is significantly effective in the treatment of cancers and may lead to reduction of the potential toxicity of both AAVtk/GCV and γ-ray.

In Vivo Tumor Formation From Primate Embryonic Stem Cells

To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be particularly useful. In this chapter, we describe an example of this model. We prepared cynomolgus ES cells genetically marked with the green fluorescent protein. The cells were transplanted into the allogeneic fetus because the fetus is immunologically premature and does not induce immune responses to transplanted cells. In addition, fetal tissue compartments are rapidly expanding, presumably providing space for engraftment. At 3 mo posttransplantation, a fluorescent teratoma, obviously derived from transplanted ES cells, was found in the fetus. However, transplanted cell progeny were also detected (approx 1%) in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells as assessed by in situ polymerase chain reaction. Transplanted cynomolgus ES cells can engraft in allogeneic fetuses. The cells will, however, form a tumor if they leak into an improper space, such as the thoracic cavity.

Adeno-associated virus vectors for gene transfer to the brain

Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinsons disease.