Doctor of Philosophy, Osaka Prefectural University (2017)
Doctor of Veterinary Medicine, Osaka Prefectural University (2013)
Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector
STEM CELLS AND DEVELOPMENT
Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.
View details for PubMedID 30215317
Generation of Canine Induced Extraembryonic Endoderm-Like Cell Line That Forms Both Extraembryonic and Embryonic Endoderm Derivatives
STEM CELLS AND DEVELOPMENT
2017; 26 (15): 1111–20
Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.
View details for DOI 10.1089/scd.2017.0026
View details for Web of Science ID 000406460500004
View details for PubMedID 28474540
Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions
MOLECULAR REPRODUCTION AND DEVELOPMENT
2017; 84 (4): 329–39
Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.
View details for DOI 10.1002/mrd.22789
View details for Web of Science ID 000400592200006
View details for PubMedID 28240438
Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs
JOURNAL OF VETERINARY MEDICAL SCIENCE
2015; 77 (7): 771–75
Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.
View details for DOI 10.1292/jvms.14-0558
View details for Web of Science ID 000358816000002
View details for PubMedID 25715707
View details for PubMedCentralID PMC4527497
Enhancement of anti-tumor immune responses by transfection of IFN gamma gene into tumor using a novel type synthetic vector
VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY
2014; 162 (1-2): 59–64
The existence of Th1 responses in a tumor microenvironment elicits a better prognosis for the patients. Transfection of Th1 polarizing cytokines, such as IFNγ, into tumor cells is an effective way to set up an appropriate microenvironment. Using a novel type synthetic vector composed of polyamidoamine dendrons, we transfected canine IFNγ gene into canine tumor cell lines, and examined direct and indirect effects of dendritic cells (DCs) against tumor growth in vitro. A cloned canine IFNγ gene expressed functional protein that induces maturation of DCs. When the canine IFNγ gene was transfected into canine tumor cell lines using the synthetic vector, most cells secreted canine IFNγ. Secretion of IFNγ reduced with time, but was maintained for 48h. DCs incubated with the IFNγ-transfected tumor cells exhibited greater suppressive activity and induced significantly higher cytotoxic activity against the tumor cells, relative to those incubated with untransfected tumor cells and comparable dose of IFNγ. Successful transfection of IFNγ by the synthetic vector efficiently enhanced the anti-tumor immune function of DCs, and sets up a suitable microenvironment for improvement in tumor therapy.
View details for DOI 10.1016/j.vetimm.2014.08.016
View details for Web of Science ID 000345202800007
View details for PubMedID 25239100
Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells.
Stem cells and development
Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1 and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific megakaryocyte and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate (ADP) or thrombin, bound to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into megakaryocytes and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.
View details for DOI 10.1089/scd.2012.0701
View details for PubMedID 23409943