Masaki Kawamata

Systemic Delivery of Synthetic MicroRNA-16 Inhibits the Growth of Metastatic Prostate Tumors via Downregulation of Multiple Cell-cycle Genes

Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.

IFATS Collection: In Vivo Therapeutic Potential of Human Adipose Tissue Mesenchymal Stem Cells After Transplantation into Mice with Liver Injury

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell‐based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue‐derived mesenchymal stem cells (AT‐MSCs), so‐called adipose‐derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT‐MSCs by their transplantation into nude mice with CCl4‐caused liver injury. We observed that after transplantation, AT‐MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic‐pyruvic transaminase, and glutamic‐oxaloacetic transaminase concentrations returned to a nearly normal level after AT‐MSC transplantation. These results raised the question of how AT‐MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT‐MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM‐MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT‐MSCs secrete interleukin 1 receptor α (IL‐1Rα), IL‐6, IL‐8, granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM‐MSCs and NHDFs. Thus, our findings suggest that AT‐MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment.

Generation of genetically modified rats from embryonic stem cells

At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/enhancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rho-associated kinase, mitogen-activated protein kinase, TGF-β, and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germline transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases.

New aspects of oxytocin receptor function revealed by knockout mice: sociosexual behaviour and control of energy balance

To further define the function of the oxytocin receptor (OXTR) in vivo, we generated mice deficient in the Oxtr gene (Oxtr-/-). Oxtr-/- mice had no obvious deficits in fertility or sexual behaviour, but displayed several aberrations in social behaviours, including male aggression, and mother-offspring interaction. In addition, they showed novel physiological defects including obesity, and dysfunction in body temperature control when exposed to cold. We review here our new findings with Oxtr-/- mice, and introduce newly generated Oxtr-Venus knockin mice as a potential tool for examining molecular physiology of Oxtr-neurons.

Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases.

Temperature-dependent change of serological specificity of Candida albicans NIH A-207 cells cultured in yeast extract-added Sabouraud liquid medium: disappearance of surface antigenic factors 4, 5, and 6 at high temperature

The cells of Candida albicans NIH A‐207 strain (A‐strain) cultivated in YSLM at high temperatures (37 and 40°C) did not undergo agglutination with the factor sera 4, 5, and 6 in a commercially available factor serum kit, ‘Candida Check’, and formed a grape‐like shape. The mannans isolated from the cells had lost their reactivity against the factor sera in ELISA. It was also revealed by 1H NMR analysis that the mannans contained neither a phosphate group nor a β‐1,2‐linked mannopyranose unit, although these mannans increased the non‐reducing terminal α‐1,3‐linked mannopyranose unit. The cells and the mannans prepared by cultivation at such high temperatures followed by 27°C in the same medium entirely recovered the reactivity with the factor sera.

Mice deficient in Dmrt7 show infertility with spermatogenic arrest at pachytene stage

Genes including DM domain regulate sexual development in diverse metazoan phyla. One of these genes, Dmrt7, was expressed only in testes of adult mice. To determine the role of Dmrt7 in mice, we generated Dmrt7‐knockout mice (Dmrt7 −/−). Although the Dmrt7 −/− showed normal growth, null males were infertile. No sperm was detected in the epididymis of Dmrt7 −/− adult males. Absence of spermatids in a histological analysis, decreased expression of Ccna1 mRNA and the accumulation of SCP3‐positive spermatocytes showed the arrest of spermatogenesis at the pachytene stage in the Dmrt7‐knockout mice.

Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans

In the non-pregnant mouse myometrium, both arginine vasopressin and oxytocin induced contractions (pD(2)=8.55+/-0.13 and 9.23+/-0.09, respectively). The effect of oxytocin was the most potent, while the maximum contractions induced by these two peptides were almost of the same magnitude. Both vasopressin- and oxytocin-induced contractions were strongly inhibited by an oxytocin receptor antagonist, CL-12-42 (d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)(9)]OVT), and weakly inhibited by a vasopressin V(1a) receptor antagonist, SR49059 ((2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide). Similar results were obtained in the pregnant mouse myometrium. These results suggest that not only oxytocin- but also vasopressin-induced contraction is mediated by the activation of oxytocin receptors in the mouse myometrium. A reverse transcription polymerase chain reaction study failed to reveal mRNA of the vasopressin V(1a) receptor in the mouse myometrium. In contrast, in the non-pregnant human myometrium, vasopressin-induced contraction was inhibited by SR49059. Oxytocin showed no effect on the myometrium. These results suggest that there are significant differences in the functional receptors and contractile responses to vasopressin and oxytocin in the human and mouse uteri.

Establishment of embryonic stem cells from rat blastocysts.

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. Their embryonic stem (ES) cells, after differentiation into each tissue or organ, are applied in regenerative medicine, which enables examination of the effects of drugs for various diseases. Knockout rats will also provide a suitable model system for many human diseases and a great amount of new insights into gene functions, which have not been revealed by knockout mice. In 2008, we experienced the worlds first success in establishing rat ES cells with chimeric contribution. Following on the heels of our report, others reported the establishment of rat ES cells that could complete a germline transmission. Recent studies on rat as well as mouse ES cells suggest that modifications of signal inhibitors and serum in the medium are critical for the maintenance of the pluripotency of ES cells. In this chapter, we discuss techniques for the successful establishment and maintenance of rat ES cells.

Male-Specific Function of Dmrt7 by Sexually Dimorphic Translation in Mouse Testis

Dmrt7 is known to be an essential gene for spermatogenesis but not for oogenesis despite mRNA expression in both testis and ovary. In this study, we examined further expression of Dmrt7 transcript and protein. Northern blot and RT-PCR analysis revealed that there was an alternative splicing variant possessing the entire sequence of intron 1 in adult testis (intron 1 variant), in addition to the mature form of mRNA. In fetal ovary, the intron 1 variant was not expressed whereas the fully spliced form of mRNA was expressed. Immunohistochemical analyses demonstrated that DMRT7 protein was present only in spermatocytes of adult testis but not in fetal ovary. In situ hybridization analyses revealed that the fully spliced form of Dmrt7 mRNA as well as the intron 1 variant were expressed in spermatogonia, spermatids and Sertoli cells in addition to spermatocytes. We also found that poly(A) tails of Dmrt7 mRNA underwent modification of its length from 70 to 440 bp long. Unlike Arbp mRNA, the size variation of poly(A) tails was observed in immature testis in which spermatids were absent. In this study, we demonstrated that Dmrt7 had unique sexually dimorphic expression patterns in transcripts that associated with spermatocyte-specific translation, but not in ovary.