Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

Atypical hypoadrenocorticism with intact zona glomerulosa of the adrenal cortex after long-term observation: a case report of a dog.

An 8-year-old intact male pointer presented with lethargy and hypoalbuminemia. On abdominal ultrasonography, both adrenal glands were reduced in thickness. Based on the ACTH stimulation test results and the absence of electrolyte abnormalities, the dog was diagnosed with atypical hypoadrenocorticism. After treatment with low-dose prednisolone, his general condition improved, and blood tests normalized. The dog died 818 days later, and a complete autopsy was performed. Histologically, the architecture of the zonae fasciculata and reticularis was disrupted in both adrenal glands; however, the zona glomerulosa remained relatively normal. In summary, in this study, we detailed the pathological presentation of atypical hypoadrenocorticism without electrolyte abnormalities.

Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A.

Introduction: Endoderm-derived organs support indispensable functions in the body. Pluripotent stem cells can generate endoderm-derived cells or tissues and have excellent therapeutic potential to replace the functions of endodermal tissues. However, there is no viable method to induce endodermal precursor cells, definitive endoderm (DE), from canine induced pluripotent stem cells (ciPSCs). Methods: A ciPSC line was used in this study. In order to induce DE, ciPSCs were cultured with high dose activin A and fetal bovine serum. We considered the optimal differentiation period and starting cell density. Next, to reduce the remaining undifferentiated cells and improve the DE induction efficiency, DE was induced from 3D cell aggregates with knockout serum replacement instead of fetal bovine serum. Finally, hepatic and pancreatic induction were performed to investigate whether DE could differentiate into downstream lineages. Results: After differentiation, some cells expressed the DE markers FOXA2 and SOX17. DE induction period and starting cell density were found to be important for efficient DE induction. However, some cells remained undifferentiated even after optimization of cell density and culture period. Cell differentiation under 3D culture conditions reduced undifferentiated cells and the replacement of fetal bovine serum with knockout serum replacement improved the DE induction efficiency. After hepatic and pancreatic induction, cells expressed some early hepatic and pancreatic markers. Conclusions: A ciPSC line was successfully differentiated to DE efficiently using a high dose of activin A with knockout serum replacement under 3D cell culture conditions. We believe that this study will be fundamental to achieving the generation of canine endodermal tissues from ciPSCs.

Mesenteric lymph node abscesses due to Escherichia coli in a cat.

A 3-year-old, castrated male mixed-breed cat presented with an almost 2-year history of chronic loose stools. On radiography and ultrasound examination, there were two masses in the centre of the abdomen. Contrast-enhanced computed tomography revealed that the masses were enlarged mesenteric lymph nodes with fluid accumulation. Percutaneous lesion drainage yielded pus-like fluid. Fluid cytology revealed numerous neutrophils and Gram-negative rods. Pus culture identified Escherichia coli as the causative organism. Consequently, mesenteric lymph node abscesses were definitively diagnosed. Since computed tomography showed that the abscesses adhered to the surrounding tissues, it was difficult to remove them surgically. With drainage and antimicrobial therapy, the mesenteric lymph nodes gradually decreased in size. However, loose stools persisted. The cat's diet was changed to a hydrolysed diet, and the clinical symptoms improved, suggesting food-responsive enteropathy. This may be an underlying disease of lymph node abscesses. Lymph node abscesses limited to the mesenteric lymph nodes rarely occur in veterinary medicine, and this is the first report in cats.

Generation of molecular-targeting helix-loop-helix peptides for inhibition of the interaction between cytotoxic T-lymphocyte-associated protein 4 and B7 in the dog.

Blocking the interaction between CD28 and B7 by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a potent immune checkpoint that prevents damage to host tissues from excessive immune responses. However, it also significantly diminishes immune responses against cancers and allows cancer cell growth. This study found that recombinant (r) human (h) CTLA-4 specifically binds to canine dendritic cells (DCs) and suppresses the responses of canine T cells to allogeneic DCs. ERY2-4, a peptide targeting rhCTLA-4 selected from a yeast-displayed library of helix-loop-helix (HLH) peptides and improved to have a binding affinity to rhCTLA-4 as strong as that of rhB7, inhibited the binding of rhCTLA-4 to canine DCs. Furthermore, the targeting peptide significantly enhanced the response of canine T cells to allogeneic DCs. These results suggest that the CTLA-4-targeting peptide enhances canine T cell activity by blocking the interaction between canine CTLA-4 on T cells and canine B7 on DCs. This study demonstrates the generation of a new type of immune checkpoint inhibitor, which may be applicable to cancer therapy in dogs.

New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix-Loop-Helix Peptide to Bind VEGF for Tumor Growth Inhibition.

As a small affinity molecule to serve as an alternative to antibodies, we have developed a conformationally constrained peptide with a de novo designed helix-loop-helix (HLH) scaffold. To evaluate its potential for biomedical applications, we performed directed evolution of HLH peptides to obtain an inhibitor for vascular endothelial growth factor-A (VEGF). A phage-displayed library of HLH peptides was constructed and screened against VEGF, giving the peptide VS42 that inhibits the VEGF/VEGF receptor-2 interaction (IC50 = 210 nM), which was further improved by in vitro affinity maturation using a yeast-displayed library. An identified HLH peptide, VS42-LR3, exhibited improved inhibitory activity (IC50 = 37 nM), high thermal stability, and excellent resistance against chemical denaturation. In biological activity tests, the HLH peptide was found to block VEGF-induced proliferation of human umbilical vein endothelial cells and suppress tumor growth in a murine xenograft model of human colorectal cancer.

Potent adjuvant effect elicited for tumor immunotherapy by a liposome conjugated pH-sensitive polymer and dendritic cell-targeting Toll-like-receptor ligand.

The generation of DCs with augmented functions is a strategy for obtaining satisfactory clinical outcomes in tumor immunotherapy. We developed a novel synthetic adjuvant comprising a liposome conjugated with a DC-targeting Toll-like-receptor ligand and a pH-sensitive polymer for augmenting cross-presentation. In an in vitro study using mouse DCs, these liposomes were selectively incorporated into DCs, significantly enhanced DC function and activated immune responses to present an epitope of the incorporated antigen on the major histocompatibility complex class I molecules. Immunization of mice with liposomes encapsulating a tumor antigen significantly enhanced antigen-specific cytotoxicity. In tumor-bearing mice, vaccination with liposomes encapsulating a tumor antigen elicited complete tumor remission. Furthermore, vaccination significantly enhanced cytotoxicity, targeting not only the vaccinated antigen but also the other antigens of the tumor cell. These results indicate that liposomes are an ideal adjuvant to develop DCs with considerably high potential to elicit antigen-specific immune responses; they are a promising tool for cancer therapy with neoantigen vaccination.

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro.

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.

Quality of life-improving effect of autologous ex vivo expanded cytotoxic and opioid-producing lymphocytes for dogs with cancers.

Activated lymphocyte therapy is one of the immunotherapies for cancer patients that is expected to prolong life without any adverse effects and maintain satisfactory quality of life (QOL). However, the objective assessment and maintenance of a standardized evaluation of QOL are not easy. We aimed to evaluate activated autologous lymphocyte therapy for cancer dogs using the characteristics of the cultured cells and QOL as perceived by owners. In in vitro experiments, peripheral blood mononuclear cells (PBMCs) collected from healthy dogs were stimulated using anti-CD3 antibody and recombinant interleukin-2 under a closed system. The number of CD4+ and CD8+ T lymphocytes in the cultured cells was higher than that of PBMCs (P < 0.05). Natural killer activity, proenkephalin (known as the precursor of endogenous opioids) and interferon-γ mRNA in activated lymphocytes were significantly higher than in PBMCs (P < 0.05). Met-enkephalin was detected in activated lymphocytes. QOL of 58 dogs afflicted with common types of cancers in humans increased after every administration of activated lymphocyte therapy (P < 0.05). Overall, these results indicated that activated lymphocyte therapy could have beneficial effects on QOL in dogs with cancers. This was objectively evaluated and this improvement was related to presence of opioid-producing lymphocytes.

Application of Zwitterionic Polymer Hydrogels to Optical Tissue Clearing for 3D Fluorescence Imaging.

Zwitterionic polymers have both anion and cation groups in the side chain and have been used in various biomedical applications because of the unique properties. In this study, zwitterionic polymer hydrogels are applied to optical tissue clearing for 3D fluorescence imaging. Polyacrylamide hydrogels have been employed in Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue-hYdrogel method. Zwitterionic polymer hydrogels are produced using zwitterionic monomers, such as 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate (DAPS) and 2-methacryloyloxyethyl phosphorylcholine (MPC), and crosslinkers. The hydrogels made from poly(DAPS-co-acrylamide) and MPC homopolymers afford the most transparent tumor tissues. However, the tissues cleared using DAPS copolymers-containing hydrogels became turbid in a refractive index-matching solution, which are unable to obtain clear 3D fluorescence images. In contrast, the 3D fluorescence imaging is achieved in the MPC polymer-treated 2-mm-thick brain slices after immunostaining. The 3D fluorescence imaging of lung metastasis that is cleared by the MPC hydrogel to demonstrate the possible application to cancer diagnosis is performed. The results indicate the increased potentials of zwitterionic polymer hydrogels, especially MPC polymer hydrogels, in biomedical applications.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

Microbial Antigen-Presenting Extracellular Vesicles Derived from Genetically Modified Tumor Cells Promote Antitumor Activity of Dendritic Cells.

Tumor-derived extracellular vesicles (EVs), as tumor vaccines, carry tumor-associated antigens (TAAs), and were expected to transfer TAAs to antigen-presenting cells. However, treatment with tumor-derived EVs exhibited no obvious antitumor effect on the established tumors, likely due to their immuno-suppressive functions, and also to the poor immunogenicity of TAAs. In order to improve the immune stimulating properties, EVs expressing a highly immunogenic bacterial antigen, 6 kDa early secretory antigenic target (ESAT-6), from Mycobacterium tuberculosis were prepared by genetically modifying the parent tumor cells with a plasmid coding for ESAT-6. Cultured B16 tumor cells were transfected with a ternary complex system consisting of pDNA, polyethylenimine (PEI), and chondroitin sulfate. The cells that were transfected with the ternary complex secreted EVs with a higher number of ESAT-6 epitopes than those transfected by a conventional DNA/PEI binary complex, due to the low cytotoxicity, and durable high expression efficiency of the ternary complex systems. The EVs presenting the ESAT-6 epitope (ESAT-EV) were collected and explored as immune modulatory agents. Dendritic cells (DCs) were differentiated from mouse bone marrow cells and incubated with ESAT-EV. After incubating with the EVs for one day, the DCs expressed a significantly higher level of DC maturation marker, CD86. The DCs treated with ESAT-EV showed a significantly improved antitumor activity in tumor-bearing mice.

Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Canine mesenchymal stromal cell-conditioned medium promotes survival and neurite outgrowth of neural stem cells.

We examined the paracrine action of canine mesenchymal stromal cells (MSCs) derived from bone marrow on the survival and differentiation of neural stem cells (NSCs) in vitro. MSCs were collected from the proximal end of the diaphysis of femur of healthy beagle dogs. The 70-80% confluent MSCs were re-fed with serum-free DMEM. The MSCs were incubated for 48 hr and the supernatant was collected as the conditioned medium (MSC-CM). The survival rate of NSCs in MSC-CM was significantly greater than in the medium without MSC-CM. The percentage of differentiated neurons and neurite length in MSC-CM was also significantly higher than in the medium without MSC-CM. These results suggested that canine MSC-CM promotes stem cell survival and neural differentiation of NSCs.

Development of feline embryos produced using freeze-dried sperm.

Freeze drying has been developed as a new sperm preservation method that eliminates the necessity of using liquid nitrogen. An advantage of freeze-dried sperm is that it can be stored at 4 °C and transported at room temperature. To develop assisted reproductive techniques (ARTs) for domestic cats, we evaluated the effect of the freeze-dry procedure on cat sperm DNA by analyzing DNA integrity (experiment 1) and by generating cat embryos using freeze-dried sperm that had been preserved for several months (experiment 2). In experiment 1, the rate of DNA damage to freeze-dried sperm was not significantly different than that of sperm cryopreserved with liquid nitrogen (P > 0.05). In experiment 2, the proportions of cleaved embryos, morulae, and blastocysts and the cell number of blastocysts did not differ between experimental groups in which fresh sperm and freeze-dried sperm were used (P > 0.05). In addition, we generated feline blastocysts using freeze-dried sperm stored for 1-5 months. These results support an expansion of the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.