A passage-free, simplified, and scalable novel method for iPSC generation in three-dimensional culture.

Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.

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.

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.

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.

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.

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.

Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell-based immunotherapy.

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 interferon-gamma, 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.

Development of feline embryos produced by Piezo-actuated intracytoplasmic sperm injection of elongated spermatids.

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector.

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.

Antibodies against swine influenza virus neutralize the pandemic influenza virus A/H1N1.

The most effective method for the prevention of influenza infection would be prophylaxis with a safe and effective vaccine and anti-viral materials. After vaccination, neutralizing antibodies are generated by plasma cells following various immune responses, thus resulting in protection against an infectious agent expressing the same antigens. However, in the case of novel or unknown pathogens, the onset of immune responses is occasionally delayed, thus resulting in considerable morbidity and mortality. Antibodies are therefore considered to play an important role in preventing infectious diseases. Furthermore, antibodies are used for additional purposes, including diagnosis and immunotherapy. In the beginning of spring 2009, an outbreak of influenza in North America was caused by a novel strain of influenza virus, designated pandemic influenza A/H1N1 2009. Initially, most people had low immunity against this pathogen, resulting in the worldwide spread of the infection to produce a so-called 'pandemic'. We herein report the generation of 'immunoglobulin yolk (IgY)' neutralizing antibodies against the pandemic influenza virus A/H1N1 from ostrich eggs immunized with a swine influenza virus vaccine strain. Using this simple method, a large amount of specific antibody against the influenza virus was produced by one female ostrich. An enzyme-linked immunosorbant assay and immunocytochemistry indicated that the IgY from the immunized ostrich eggs possessed strong cross-reactivity to the pandemic influenza virus A/H1N1 2009, as well as to the swine influenza virus. Moreover, the hemaggregation activities of the erythrocytes induced by pandemic influenza A/H1N1 virus were inhibited by the ostrich antibodies generated by swine virus immunization. In addition, the cytopathological effects on MDCK cells of infection with pandemic virus were clearly inhibited in co-cultures with the antibodies, indicating the neutralizing of viral infectivity in the cells. In conclusion, we have succeeded in the mass production of neutralizing antibodies against pandemic influenza virus A/H1N1 2009 using ostrich eggs immunized with swine influenza virus antigens. This enables the cost-effective production of effective antibodies, which could be applied to facial masks and air-conditioning filters in order to prevent populations from acquiring pandemic influenza virus A/H1N1.

Protection from avian influenza H5N1 virus infection with antibody-impregnated filters.

There is worldwide concern over the possibility of a new influenza pandemic originating from the highly pathogenic avian H5N1 influenza viruses. We herein demonstrate that functional air filters impregnated with ostrich antibodies against the hemagglutinin of the H5N1 virus protect chickens from death by H5N1 transmission. These results suggest that the use of ostrich antibody-impregnated filters might be a powerful way to prevent the transmission of H5N1.

Expression of SC1, a cell adhesion molecule, promotes the metastatic activities of the Gallus gallus lymphoblastoid cell line MDCC-MSB1 derived from Marek's disease.

SC1 is an immunoglobulin superfamily cell adhesion molecule purified from the Gallus gallus spinal cord. SC1 is present in embryonic tissues and plays a role in chick development through its cell adhesive property. Interestingly, increased SC1 expression is observed in some sporadic tumours of the chicken, including Marek's disease-induced lymphomas and in nephroblastomas. To elucidate the possible functions of SC1 in tumour progression in the chicken, SC1 cDNA was introduced into the endogenous SC1-negative Marek's disease-derived chicken lymphoblastoid cell line MDCC-MSB1, and subsequently the metastatic potentials of these cell lines were analysed. The in vitro analyses revealed that the SC1-transfected MDCC-MSB1 cells were enhanced in their adhesive and migratory activities in the presence of the SC1 proteins. In addition, the metastatic potential of the SC1-transfected MDCC-MSB1 cells to the lung was enhanced after intravenous implantation into chickens. These findings suggest that the expression of SC1 contributes to the malignancy and metastatic properties of chicken Marek's disease-induced lymphomas.

Ostrich produce cross-reactive neutralization antibodies against pandemic influenza virus A/H1N1 following immunization with a seasonal influenza vaccine.

An outbreak of influenza in 2009 was found to be caused by a novel strain of influenza virus designated as pandemic influenza A/H1N1 2009. Vaccination with recent seasonal influenza vaccines induced little or no cross-reactive antibody response to the pandemic influenza virus A/H1N1 2009 in any age group in human populations. Accordingly, most people had low immunity against this pathogen, thus resulting in the worldwide spread of the infection to produce a so-called 'pandemic'. This report presents the important finding that ostrich eggs generate cross-reactive antibodies to the pandemic influenza virus A/H1N1 following immunization of female ostrich with a seasonal influenza vaccine. This simple method produced a large amount of antibodies against influenza viruses by one female ostrich. An enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry indicated that the ostrich antibodies possessed strong cross-reactivity to the pandemic A/H1N1 as well as to the seasonal A/H1N1, A/H3N2 and B viruses. The hemaggregation activities of erythrocytes induced by this pandemic strain were also inhibited by the ostrich antibodies. In addition, the cytopathological effects of infection with a pandemic virus on MDCK cells were clearly inhibited in co-cultures with the ostrich antibodies, thereby indicating the neutralization of viral infectivity in the cells. In conclusion, cross-reactive neutralization antibodies against pandemic influenza virus A/H1N1 2009 were successfully generated in ostrich eggs produced by females immunized with seasonal influenza viral vaccine.

Involvement of gicerin, a cell adhesion molecule, in the portal metastasis of rat colorectal adenocarcinoma cells.

Gicerin, an Ig-superfamily cell adhesion molecule, has homophilic adhesion activity, thus leading to the formation of gicerin aggregates. Gicerin is highly expressed in various embryonic tissues, and it contributes to development through its adhesive activities. In contrast, the expression of the protein is limited to the muscular tissues and endothelial cells in the mature animals. In the liver, gicerin is constitutively expressed in sinusoidal endothelial cells. Interestingly, an overexpression of gicerin is found in a variety of tumors and may play a role in tumorigenesis. Previously, up-regulated expression of the gicerin protein was found in some sporadic cases of chicken colorectal adenocarcinomas and their hepatic metastasized lesions. In the present study, gicerin cDNA was introduced into endogenous gicerin negative ACL-15 cells, a rat colon adenocarcinoma cell line. The cells were subsequently evaluated for changes in their metastatic potentials in order to elucidate the possible role of gicerin in the hepatic metastasis of colorectal adenocarcinomas. The stable overexpression of gicerin in the cells enhanced the self-aggregation and migratory activities on the protein compared with the mock-transfectants. In addition, the gicerin- transfectants had enhanced metastatic potential to the liver compared with mock-transfected cells after implantation into the ileocolic vein of the cognate rats. These results suggest that gicerin might promote the interaction of tumor cells with a hepatic endothelium, thus leading to the hepatic metastasis of colon adenocarcinomas.

Involvement of gicerin, a cell adhesion molecule, in a dermal autograft chicken model.

Gicerin is a cell adhesion molecule in the immunoglobulin superfamily. This molecule has homophilic and heterophilic adhesive activities, binding to the neurite out-growth factor (NOF). We have previously reported that gicerin plays an important role in the development and regeneration as well as in the metastasis of tumors through its adhesive activities, mediating cell-cell and/or cell-extracellular matrix interactions. In this study, we investigated the involvement of gicerin in a dermal autograft chicken model. Gicerin and NOF were transiently present in the regenerating epithelia after the dermal graft transplantation. The treatment with an anti-gicerin polyclonal antibody, by placing drops onto the wounds, inhibited the adhesiveness of the grafts to the marginal skin. The chimeric protein of gicerin-IgG, gicerin-Fc, and NOF proteins promoted the regeneration of the grafts. These findings suggest the potential function of gicerin in dermal autografts, and gicerin and NOF proteins could help clinical improvement after transplantations.