Hajime Ishii

Transplantation of human neural stem cells for spinal cord injury in primates

Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro‐expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham‐operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham‐operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham‐operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.

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.

Establishment of graded spinal cord injury model in a nonhuman primate: The common marmoset

Most previous studies on spinal cord injury (SCI) have used rodent models. Direct extrapolation of the results obtained in rodents to clinical cases is difficult, however, because of neurofunctional and anatomic differences between rodents and primates. In the present study, the development of histopathologic changes and functional deficits were assessed quantitatively after mild, moderate, and severe spinal cord contusive injuries in common marmosets. Contusive SCI was induced by dropping one of three different weights (15, 17, or 20 g) at the C5 level from a height of 50 mm. Serial magnetic resonance images showed significant differences in the intramedullary T1 low signal and T2 high signal areas among the three groups. Quantitative histologic analyses revealed that the number of motor neurons, the myelinated areas, and the amounts of corticospinal tract fibers decreased significantly as the injury increased in severity. Motor functions were evaluated using the following tests: original behavioral scoring scale, measurements of spontaneous motor activity, bar grip test, and cage‐climbing test. Significant differences in all test results were observed among the three groups. Spontaneous motor activities at 10 weeks after injury were closely correlated with the residual myelinated area at the lesion epicenter. The establishment of a reliable nonhuman primate model for SCI with objective functional evaluation methods should become an essential tool for future SCI treatment studies. Quantitative behavioral and histopathologic analyses enabled three distinct grades of injury severity (15‐g, 17‐g, and 20‐g groups) to be characterized with heavier weights producing more serious injuries, and relatively constant behavioral and histopathologic outcomes.

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.

Pharmacokinetics of CPT-11 in rhesus monkeys

Purpose: To examine the pharmacokinetic relationships between humans and monkeys, we studied the disposition of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) and its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), in rhesus monkeys. Methods: CPT-11 was administered to a total of six monkeys at doses of 3, 7, 15 and 25u2009mg/kg by intravenous infusion for 10u2009min and plasma concentrations and pharmacokinetic parameters of CPT-11 determined. Results: Maximum plasma concentrations at 25u2009mg/kg reached around 10u2009000u2009ng/ml, and dropped to 500u2009ng/ml in 8u2009h. Plasma concentrations of SN-38 remained between 2 and 10u2009ng/ml. Mean values of systemic clearance, mean residence time and distribution volume at steady state, the major pharmacokinetic parameters for CPT-11, were 13.3u2009(ml/min per kg), 192u2009(min) and 2553u2009(ml/kg), respectively. The initial plasma concentration ratio of lactone to total CPT-11, 76%, declined to about 20% within 75u2009min, and the final ratio was about 40% at 8u2009h; the initial ratio of SN-38 was 72%, dropped to 34% within 70u2009min and finally recovered to 55% at 8u2009h. Conclusion: Comparison with human data revealed that systemic clearances of CPT-11 and the maximum AUC of SN-38 were not as different between humans and monkeys as between humans and mice, but the metabolic conversion of CPT-11 into SN-38 in monkeys was significantly lower than in humans.

Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset (Callithrix jacchus)

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

Vocalization pattern analysis in the common marmosets (Callithrix jacchus);Novel method to classify emotional states of animals

s / Neuroscience Research 68S (2010) e335–e446 e427 P3-p08 Transplantation of human iPS cell-derived neurospheres for the treatment of spinal cord injury in NOD-scid mice Satoshi Nori 1 , Yohei Okada 2, Osahiko Tsuji 1, Yuichiro Takahashi 1, Kanehiro Fujiyoshi 1, Akimasa Yasuda 1, Yoshiomi Kobayashi 1, Yoshiaki Toyama 1, Shinya Yamanaka 3, Masaya Nakamura 1, Hideyuki Okano 2 1 Department of Orthop Surg, Keio Univ, Tokyo, Japan 2 Department of Physiol, Keio Univ, Tokyo, Japan 3 CiRA, Kyoto Univ, Kyoto, Japan Induced pluripotent stem (iPS) cells have the potential to resolve the ethical issues and immunological rejection associated with embryonic stem (ES) cells. Recently, we have reported the effectiveness of transplantation of mouse iPS cell-derived neurospheres (iPS-NS) for spinal cord injury (SCI) in mice. In the present study, we performed the transplantation of human iPS-NS into injured spinal cord of NOD-scid mice to examine their therapeutic potential and safeness as a cell source of transplantation for SCI. Human iPS cells had been generated from adult human dermal fibroblasts by retroviral transduction of four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc: c-Myc(+)iPS) or three transcription factors (Oct3/4, Sox2 and Klf4: c-Myc()iPS) (Takahashi et al., 2007 Cell). We had established the directed neural differentiation of human iPS cells. Human iPS-NS were mainly differentiated into neurons and less glial cells in vitro. Adult female NOD-scid mice were used in this study. After laminectomy, contusive spinal cord injury was induced at Th10 level using IH impactor. Nine days after the injury, the mice were to receive randomized c-Myc(+) iPS-NS, c-Myc(-)iPS-NS or PBS. Motor functions in the hindlimbs were assessed by BMS and Rotarod treadmill. Grafted human iPS-NS survived and differentiated into neurons, astrocytes and less oligodendrocytes in the injured spinal cord. We found that the most of human iPS cell-derived neurons were GABAergic neurons, and also found the synapse formation between human iPS cell-derived neurons and host neurons. It is known that angiogenesis has the positive effect for tissue repair in the injured spinal cord, and we observed the transplantation of human iPSNS promoted angiogenesis. As a result, the human iPS-NS groups exhibited significantly better functional recovery than the control group.Further investigation is being performed for the effectiveness and the safety issues about the transplantation of human iPS-NS for SCI. doi:10.1016/j.neures.2010.07.1892 P3-p09 Aquaporin-4 plays a pivotal role in developing brain edema after traumatic brain injury in alcohol-treated rats Ryuichi Katada , Keisuke Mizuo, Shunichiro Okazaki, Kenji Tateda, Satoshi Watanabe, Hiroshi Matsumoto Department of Legal Medicine and Molecular Alcohology, Sapporo Medical University School of Medicine Ethanol consumption affects morbidity and motrtality after traumatic brain injury (TBI). We previously reported that prior ethanol injection exacerbated mortality by accelerating brain edema after TBI and that N-acetyl cysteine (a precursor of glutathione) improved brain edema augmentation after TBI under ethanol consumption (Katada et al, 2009. Journal of Neurotrauma). On the other hand, it was reported that aquaporin-4 (AQP4) is one of water channels expressed abundantly in brain. We hypothesized that AQP4 contributes to brain edema formation. To elucidate the role of AQP4 in augmentation of brain edema after TBI under ethanol consumption, we used three rat models divided into saline group, ethanol group andDL-buthionine(S,R)-sulfoximine (BSO) (a glutathione depletor) group. Ethanol (3 g/kg body weight) and BSO (100 mg/kg body weight) were injected i.p. before TBI. TBI was performed by weight-drop device method as described previously. We assessed brain edema by Magnetic Resonance Imaging (7.0 tesla), and examined AQP4 expression by western blotting and immunohistochemistory in rat brain. Both ethanol and BSO increased AQP4 expression at 24 hr after TBI by western blotting. AQP4 was expressed in astrocyte end-feet distributed along subependymal vessels and capillary vessels at 24 hr after TBI. Ethanol pretreatment accelerated AQP4 expression. These findings indicate that accumulation of AQP4 expression plays an important role in augmentation of brain edema after TBI. The inhibition of AQP4 may improve the mobidity and mortality in brain injury patients. doi:10.1016/j.neures.2010.07.1893 P3-p10 Activation of spinal microglia contributes to paclitaxel-induced mechanical allodynia, cold allodynia and motor dysfunction Kenichiro Nagata 1,2 , Tomoyuki Inoue 1, Takayuki Yano 1, Yuta Kohro 1, Ryozo Oishi 2, Makoto Tsuda 1, Kazuhide Inoue 1 1 Dept. of Mol Sys. Pharmacol., Grad Sch. Pharmaceut., Kyushu University, Fukuoka, Japan 2 Dept. of Pharmacy, Kyushu University Hospital, Fukuoka, Japan Paclitaxel, an effective chemotherapeutic agent, is widely used for the treatment of solid tumors, including breast, ovarian and non-small cell lung cancer. However, its use is often limited because of the incidence of peripheral neuropathy, which is characterized by a sensory abnormality of extremities and motor dysfunction, but the underlying mechanisms are largely unknown. Emerging lines of evidence indicate that activated microglia in the spinal cord cause abnormality of sensory and motor function. Recently, it has been reported that paclitaxel induce activation of spinal microglia in the rat, but its role in the pathogenesis of paclitaxelinduced neuropathy is not fully elucidated. The aim of the present study is to investigate the involvement of activation of spinal microglia in the paclitaxel-induced peripheral neuropathy. Repeated administration of paclitaxel caused mechanical allodynia, cold allodynia and motor dysfunction in rats. In the dorsal horn of the L5 spinal cord of paclitaxel-treated rats, increased Iba-1 labeling (a marker of microglia) and hypertrophic morphology of microglia (such as enlarged cell body and retracted processes) were observed. In the ventral horn, activation of microglia was localized to the specific area where neuronal cells projecting muscles of lower legs are present. Repeated intrathecal administration of minocycline, which is known to inhibit activation of microglia, significantly reduced the number of Iba-1-positive cells in both the dorsal and ventral horn of the L5 spinal cord, and reversed mechanical allodynia, cold allodynia and motor dysfunction. These results suggest that activation of spinal microglia may play a crucial role in the paclitaxel-induced behavioral signs of neuropathy, and the inhibition of activating microglia may be a new therapeutic strategy for treating paclitaxel-induced neuropathy. doi:10.1016/j.neures.2010.07.1894 P3-p11 Vocalization pattern analysis in the common marmosets (Callithrix jacchus);Novel method to classify emotional states of animals Hayato Gokan 1,2 , Arata Oh-Nishi 1, Takafumi Minamimoto 1, Hajime Ishii 1, Shigeru Watanabe 2, Tetsuya Suhara 1 1 Neuroimaging, National Institute of Radiological Science, Chiba, Japan 2 Dept Psy, Univ of Keio, Tokyo In order to classify emotional states of animals, common marmosets may be useful subjects. It is well known that there is strong correlation between the patterns of vocalization and the specific behavior in the common marmosets (e.g., ?chatter? call corresponds aggressive behavior). This suggests that the vocalization patterns of marmosets may be useful to classify emotional states of the animals. Here, we collected vocalization patterns of the marmoset in the isolated environment and tried to map their corresponding emotional states. Two adult common marmosets were used in this study. Each animal was isolated in the novel cage, and habituated for 60 min for 5days. The vocalization was recorded for the first 30 min of the habituation. The number of vocalization decreased significantly after completion of habituation in comparison with the first habituation trial. The number of patterns of vocalization also decreased, so that they vocalized mainly ?phee? call, which corresponds ?calling partners?, after habituation. They often vocalized with specific pattern, ?moaning? call at the first habituation trial. This suggests that ?moaning? call reflects anxiety evoked by the novel environment. With the use of this vocalization pattern analysis, we tried a methamphetamine test in the same environment. The number of ?moaning? calls in 30 min significantly increased with methamphetamine (METH) (2mg/kg) treatment in comparison with the saline treatment, suggesting that METH makes marmoset anxiety. Taken together, vocalization patterns of marmosets may allow us to identify their emotional states with higher validity. doi:10.1016/j.neures.2010.07.1895

Novel hyperprolactinemia and hyperprolactinemic anovulation model using the cynomolgus monkey (Macaca fascicularis)

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.

Hematological Aspects of Common Marmoset Monkey Transplanted with Autologous MDR1 Gene Transduced Peripheral Blood Stem Cells

We have developed a small primate model system for human gene therapy using the common marmoset to evaluate the safety and efficacy of gene therapeutic approaches. We demonstrated that retroviral transduction of multidrug resistance gene (MDR1) into G-CSF-mobilized peripheral blood progenitor cells (PBPC) was improved by culturing PBPC with the mixed populations of bone marrow stroma cells and viral producer cells. We performed autologous PBPC transplantation using MDR1- transduced PBPC in two marmosets. The transduction efficiency to PBPC was 5.9–6.7% and provirus DNA could be detected in peripheral blood granulocytes and lymphocytes by PCR method in all gene transduced marmoset maximally up to 412 days after transplantation. MDR1-transduced autologous PBPC were tranasplanted to irradiated common marmosets and reconstituted their own hematopoiesis. Provirus MDR1 DNA could be detected in peripheral blood granulocytes and lymphocytes by PCR method in all gene transduced marmosets. Replication competent retrovirus was not detected in all marmosets by S+L− assay. Our preclinical stem/progenitor gene therapy system using marmoset is considered to be helpful for the development of human gene therapy.