Heart Failure Post-Myocardial Infarction Promotes Mammary Tumor Growth Through the NGF-TRKA Pathway.

Background: Epidemiological investigations suggest that patients with heart failure have a higher incidence of cancer; however, the causal role of cardiac disease on cancer progression remains unclear. Objectives: This study aimed to investigate the impact and underlying mechanisms of myocardial infarction (MI)-induced heart failure on tumor cell growth. Methods: We generated a syngeneic mouse model by implanting mammary tumor-derived 4T1 cells into BALB/c mice with MI resulting from ligation of the left anterior descending artery. Results: Mice with MI exhibited increased tumor volume, tumor weight, and Ki67-positive proliferative cells in the tumor tissue compared with the sham-operated mice. Furthermore, RNA sequencing analysis in the tumor tissue revealed significant enrichment of pathways related to tumor progression, particularly the PI3K-AKT pathway in the MI mice. Upregulation of tropomyosin receptor kinase A (TRKA) phosphorylation, an upstream regulator of PI3K-AKT signaling, was observed in the tumor tissue of the MI mice. We also observed elevated levels of circulating nerve growth factor (NGF), a ligand of TRKA, and increased NGF expressions in the myocardium after MI. In in vitro experiments, NGF stimulation led to increased cell proliferation, as well as phosphorylation of TRKA and AKT. Notably, inhibition of TRKA by small interfering RNA or the chemical inhibitor GW441756 effectively blocked these effects. Administration of GW441756 resulted in the suppression of tumor volume and cell proliferation in the MI mice. Conclusions: Our study demonstrates that MI promotes mammary tumor growth through the NGF-TRKA pathway. Consequently, inhibiting TRKA could represent a therapeutic strategy for breast cancer patients concurrently experiencing heart failure after MI.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Immortalization of American miniature horse-derived fibroblast by cell cycle regulator with normal karyotype.

Immortalized cells serve as a crucial research tool that capitalizes on their robust proliferative properties for functional investigations of an organism. Establishing an immortalized American miniature horse cell line could yield valuable insights into these animals' genetic and physiological characteristics and susceptibility to health issues. To date, immortalized small horse cells with normal karyotypes have not been established. In this study, we successfully established primary and immortalized fibroblast cell lines through the combined expression of human-derived mutant cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and Telomerase Reverse Transcriptase (TERT), although CDK4R24C and cyclin D1, SV40T and TERT did not result in successful immortalization. Our comparison of the properties of these immortalized cells demonstrated that K4DT immortalized cells maintain a normal karyotype. Ultimately, our findings could pave the way for the development of targeted interventions to enhance the health and well-being of American miniature horses.

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

Increased lentivirus titer using ultra expression vectors.

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

Induced pluripotent stem cells of endangered avian species.

The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation.

Correction: Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

[This corrects the article DOI: 10.1371/journal.pone.0221364.].

Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.

Mitogen-Activated Protein Kinase Activity Is Not Essential for the First Step of Nuclear Reprogramming in Bovine Somatic Cell Nuclear Transfer.

For reprogramming a somatic nucleus during mammalian cloning, metaphase of the second meiotic division (MII) oocytes has been widely used as recipient cytoplasm. High activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) is believed to accelerate the remodeling and/or reprogramming of a somatic nucleus introduced into the ooplasm by somatic cell nuclear transfer. We demonstrated previously that the first step in nuclear reprogramming is not directly regulated by MPF and MAPK because activated oocytes in which MPF activity is diminished and MAPK activity is maintained can develop to the blastocyst stage after receiving an M phase somatic nucleus in bovine cloning. In this study, our aim was to test whether MAPK activity is necessary for the first step in nuclear reprogramming and/or chromatin remodeling (phosphorylation of histone H3 at Ser3, trimethylation of histone H3 at Lys 9, and acetylation of histone H3 at Lys14) in bovine somatic cloning. We found that it was not necessary, and neither was MPF activity.

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD.

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.

Structure and functions of powerful transactivators: VP16, MyoD and FoxA.

Induced pluripotent stem cell (iPSC) technology is a promising approach for converting one type of a differentiated cell into another type of differentiated cell through a pluripotent state as an intermediate step. Recent studies, however, indicate the possibility of directly converting one cell type to another without going through a pluripotent state. This direct reprogramming approach is dependent on a combination of highly potent transcription factors for cell-type conversion, presumably skipping more physiological and multi-step differentiation processes. A trial-and-error strategy is commonly used to screen many candidate transcription factors to identify the correct combination of factors. We speculate, however, that a better understanding of the functional mechanisms of exemplary transcriptional activators will facilitate the identification of novel factor combinations capable of direct reprogramming. The purpose of this review is to critically examine the literature on three highly potent transcriptional activators: the herpes virus protein, VP16; the master regulator of skeletal muscle differentiation, MyoD and the "pioneer" factor for hepatogenesis, FoxA. We discuss the roles of their functional protein domains, interacting partners and chromatin remodeling mechanisms during gene activation to understand how these factors open the chromatin of inactive genes and reset the transcriptional pattern during cell type conversion.

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Aberrant spindle assembly checkpoint in bovine somatic cell nuclear transfer oocytes.

Nuclear, microtubular dynamics and spindle assembly checkpoint (SAC) in bovine somatic cell nuclear transfer (SCNT) oocytes receiving G1/0 or M phase somatic cell nuclei were studied. SCNT oocytes assembled microtubules, however, the spindles were structurally abnormal, including bi-, tri-polar or elongated spindles with scattered premature chromosome condensation (PCC) in G1/0 phase nuclei, and some miniature spindles with unaligned chromosomes in M phase nuclei. In contrast, demecolcine-treated SCNT oocytes formed chromosome clusters with membrane protrusion and significantly induced maturation-promoting factor (MPF) activity elevation (up to 177%) for 3 hours, indicating that first SAC at second metaphase (MII) is established upon spindle disruption in SCNT oocytes. After parthenogenetic stimuli, unlike MII oocytes which prevent exit from MII arrest with high MPF activity upon spindle disruption by second SAC, demecolcine-treated SCNT oocytes could not prevent exit from MII arrest with inactivation of MPF activities, whereas MG132-treated SCNT oocytes could persist at MII arrest, indicating that SCNT oocytes lack the ability for second SAC establishment, however, two G1/0 phase nuclei in an ooplasm restored second SAC establishment upon spindle disruption. Furthermore, the developmental potential of demecolcine-treated SCNT oocytes receiving G1/0 phase nuclei to blastocyst stage was not significantly different than untreated SCNT oocytes (29% vs 31%). These results indicate that unlike MII oocytes, SCNT oocytes have aberrant spindle morphology and SAC at MII due to insufficient SAC signals from somatic cell nuclei, thus aberrant remodeling has started immediately after somatic cell nuclear transfer and may be responsible for chromosome instability in SCNT embryos as well as the low successful efficiency of cloning.

Demecolcine-assisted enucleation for bovine cloning.

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.

Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal.

Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.