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 MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind 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 MKs 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.

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.

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.

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.

The tumor microenvironment strongly influences clinical outcomes of immunotherapy.By transfecting genes of relevant cytokines into tumor cells, we sought to manipulate the microenvironment so as to elicit activation of T helper type 1 (Th1) responses and the maturation of dendritic cells (DCs). Using a synthetic vehicle, the efficiency of in vivo transfection of GFP-cDNA into tumor cells was about 7.5% by intratumoral injection and about 11.5% by intravenous injection. Survival was significantly improved by both intratumoral and intravenous injection of the plasmid containing cDNA of interferongamma, followed by intratumoral injection of DCs presenting the tumor antigens. Also, tumor growth was inhibited by these treatments. A more significant effect on survival and tumor growth inhibition was observed following injection of the plasmid containing cDNA of CD40 ligand, which is a potent inducer of DC-maturation. Furthermore, the co-injection of both IFNγ-and CD40 ligand-encoding cDNA-plasmids, followed by DC treatment, gave rise to further marked and enhancement, including 100% survival and more than 50% complete remission. This treatment regimen elicited significant increases in mature DCs and types of cells contributing to Th1 responses, and significant decreases in immune suppressor cells in the tumor. In the spleen, the treatment significantly increased activities of tumor-specific killer and natural killer cells, but no alteration was observed in mature DCs or suppressor cells. These results indicate that transfection of these cytokine genes into tumor cells significantly alter the tumor microenvironment and improve the therapeutic results of DC-based immunotherapy. K E Y W O R D Scancer immunotherapy, CD40 ligand, gene therapy, immunity, interferon-gamma, synthetic vehicle 6 |