Translation reinitiation in c.453delC frameshift mutation of KCNH2 producing functional hERG K+ channels with mild dominant negative effect in the heterozygote patient-derived iPSC cardiomyocytes.

The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.

The antioxidant icariin protects porcine oocytes from age-related damage in vitro.

OBJECTIVE: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. METHODS: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. RESULTS: Treatment with 5 µM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. CONCLUSION: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

Impact of High-Dose Irradiation on Human iPSC-Derived Cardiomyocytes Using Multi-Electrode Arrays: Implications for the Antiarrhythmic Effects of Cardiac Radioablation.

Cardiac radioablation is emerging as an alternative option for refractory ventricular arrhythmias. However, the immediate acute effect of high-dose irradiation on human cardiomyocytes remains poorly known. We measured the electrical activities of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) upon irradiation with 0, 20, 25, 30, 40, and 50 Gy using a multi-electrode array, and cardiomyocyte function gene levels were evaluated. iPSC-CMs showed to recover their electrophysiological activities (total active electrode, spike amplitude and slope, and corrected field potential duration) within 3-6 h from the acute effects of high-dose irradiation. The beat rate immediately increased until 3 h after irradiation, but it steadily decreased afterward. Conduction velocity slowed in cells irradiated with ≥25 Gy until 6-12 h and recovered within 24 h; notably, 20 and 25 Gy-treated groups showed subsequent continuous increase. At day 7 post-irradiation, except for cTnT, cardiomyocyte function gene levels increased with increasing irradiation dose, but uniquely peaked at 25-30 Gy. Altogether, high-dose irradiation immediately and reversibly modifies the electrical conduction of cardiomyocytes. Thus, compensatory mechanisms at the cellular level may be activated after the high-dose irradiation acute effects, thereby, contributing to the immediate antiarrhythmic outcome of cardiac radioablation for refractory ventricular arrhythmias.

Antiviral activity and safety of remdesivir against SARS-CoV-2 infection in human pluripotent stem cell-derived cardiomyocytes.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.

Pioglitazone improves porcine oocyte maturation and subsequent parthenogenetic embryo development in vitro by increasing lipid metabolism.

Optimization of culture conditions is important to improve oocyte maturation and subsequent embryo development. In particular, this study analyzed the effects of increasing concentrations of PIO in the maturation medium on spindle formation and chromosome alignment, glutathione, and intracellular ROS levels and expression of selected genes related to maternal markers, apoptosis, and lipid metabolism. The percentage of oocytes displaying normal spindle formation and chromosome alignment was higher in the 1 µM PIO (1 PIO)-treated group than in the control group. The glutathione level was significantly higher in the 1 PIO-treated group than in the control group, while the reactive oxygen species level did not differ. Expression of maternal marker (MOS and GDF9), antiapoptotic (BIRC5), and lipid metabolism-related (ACADS, CPT2, SREBF1, and PPARG) genes was higher in the 1 PIO-treated group than in the control group, while expression of a proapoptotic gene (CASP3) was lower. The blastocyst formation rate and the percentage of blastocysts that reached at least the hatching stage on Days 6 and 7, and the percentage of blastocysts containing more than 128 cells were significantly higher in the 1 PIO-treated group than in the control group. These results indicate that PIO treatment during in vitro maturation improves porcine oocyte maturation and subsequent parthenogenetic embryo development mainly by enhancing lipid metabolism and antioxidant defense in oocytes.

Allicin protects porcine oocytes against damage during aging in vitro.

Allicin, a chemical component of garlic, has strong antioxidant activity and is thought to exert antiaging effects in vitro. We investigated whether allicin treatment would protect porcine oocytes and embryos from postovulatory aging mediated by apoptosis and autophagy. The rates of oocyte survival and polar body extrusion in samples treated with 1 µM allicin (1 AL) were significantly higher than in untreated samples (0 AL). In addition, 1 AL prevented defects in spindle formation and chromosome alignment, as well as decreases in the expression of maturation markers, during in vitro aging. In this study, we considered allicin to be a regulator of autophagy rather than an antioxidant or antiapoptotic agent. At the embryo level, although the cleavage rate after parthenogenetic activation was similar in all groups, the blastocyst formation rate was higher in the 1 AL group than in the 0 AL group. Our findings demonstrate that allicin effectively prevents the deterioration of porcine oocytes during aging in vitro, and could therefore be used to improve the quality of aged oocytes used in in vitro experiments.

Antioxidant hesperetin improves the quality of porcine oocytes during aging in vitro.

The citrus flavonoid hesperetin has a variety of pharmacological actions, including antioxidant, antiinflammatory, and anticancer activities. This study investigated whether hesperetin prevents aging of oocytes in vitro in which it determined the maturation of nuclear and cytoplasm and the developmental capacity of embryo by modulating the reactive oxygen species (ROS) level. Porcine oocytes were matured in vitro for 44 hr (control) and for an additional 24 hr in the presence of 0, 1, 10, 100, and 250 µM hesperetin (aging, H-1, H-10, H-100, and H-250, respectively). Although there was no difference in the rate of maturation among all the groups, both the control and H-100 groups significantly increased in the rate of cleavage and blastocyst formation compared to the aging group. The H-100 group significantly decreased ROS activity and increases the level of glutathione (GSH) and expression of the antioxidant genes (PRDX5, NFE2L, SOD1, and SOD2) compared with the aging group. The H-100 groups prevented aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase and increased the messenger RNA expression of cytoplasmic maturation factor genes (GDF9, CCNB1, BMP15, and MOS). Subsequently, both the control and H-100 groups significantly increased the total cell number and decreased the apoptosis cells at the blastocyst stage compared with aging group. The results indicate that hesperetin improves the quality of porcine oocytes by protecting them against oxidative stress during aging in vitro.

Fibroblast Growth Factor 10 Enhances the Developmental Efficiency of Somatic Cell Nuclear Transfer Embryos by Accelerating the Kinetics of Cleavage During In Vitro Maturation.

Somatic cell nuclear transfer (SCNT) is required for the generation of transgenic animals as disease models. During the in vitro development of SCNT embryos, the quality of matured oocytes is one of the major factors regulating the developmental potential of embryos. Time-lapse monitoring systems are new tools that assess the developmental capacity of embryos for use in embryo transfer. In this study, we investigated the effect of fibroblast growth factor 10 (FGF 10) on the developmental potential of SCNT embryos. After the in vitro maturation (IVM) of oocytes in IVM medium containing 10 ng/mL FGF 10 (10 F), the polar body extrusion rate was significantly higher than in the control. However, there was no difference in the percentage of fused embryos between the groups. The cleavage and blastocyst formation rates of embryos were significantly increased in the 10 F compared with the control. In addition, the total cell number was higher and the apoptotic index was lower in the 10 F than control at day 7. The messenger RNA (mRNA) expression of genes involved in apoptosis (baculoviral inhibitor of apoptosis repeat containing 5 [BIRC5] and caspase 3 [CASP3]) and development (octamer-binding transcription factor 4 [POU5F1] and sex determining region Y box 2 [SOX2]) increased after 10 F treatment. Furthermore, the kinetics of the first cleavage was faster and the percentage of embryos at cell block was significantly lower in the 10 F group than in the control. These results demonstrate that exposure of oocytes to FGF 10 during IVM promotes developmental competence.

Antioxidant ß-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro.

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant ß-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of ß-cryptoxanthin (0, 0.1, 1, 10 or 100µM). Treatment with 1µM ß-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that ß-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

Treatment of allicin improves maturation of immature oocytes and subsequent developmental ability of preimplantation embryos.

Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 µM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

Production of transgenic pig as an Alzheimer's disease model using a multi-cistronic vector system.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aß-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Intermediate Reprogramming of Mouse Skin Fibroblasts into Stem-Like Cells by Bone Morphogenetic Protein 4.

Specific transcription factors are sufficient to reprogram fully induced pluripotent stem cells or other types of cells. These findings raise the question of whether chemical molecules or proteins can replace transcription factors to alter the defined cell fate. In this study, we treated mouse skin fibroblasts (MSFs) with bone morphogenetic protein 4 (BMP4) and examined intermediate reprogramming of MSFs into stem-like cells. Putative epidermal stem cells isolated from the ventral skin epidermis of an adult mouse were used to confirm the reprogramming activity of BMP4, which increased the proliferation of these cells. After these cells formed spheroids, they were treated with BMP4 and cultured for 5 days. Following BMP4 treatment, the characteristics of these cells changed, and they expressed Oct-4 and its target transcripts Nanog, Sox2, and alkaline phosphatase. To confirm the stem cell potency of these cells, we induced their differentiation into cardiomyocytes. Stem-like cell-derived cardiomyocytes exhibited mRNA expression of cardiac mesoderm markers such as Nk2 transcription factor-related locus 5 and connexin 40, and the cardiomyocyte marker troponin T. These differentiated cells exhibited contracting masses. These results suggest that BMP4-mediated somatic stem cell reprogramming may become an alternative approach for cell therapy.

Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes.

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.

Cell Synchronization by Rapamycin Improves the Developmental Competence of Porcine SCNT Embryos.

The cell cycle stage of donor cells influences the success of somatic cell nuclear transfer (SCNT). This study investigated the effects of rapamycin treatment on synchronization of porcine fibroblasts in comparison with control and serum-starved cells, SCNT donor cell viability, and SCNT-derived embryo development. Porcine fibroblasts were treated with 0.1, 1, 10, and 100 µM rapamycin for 1 or 3 days. The proportion of cells in G0/G1 phase was significantly higher among cells treated with 1 µM rapamycin for 3 days (D3-1R) than among control and serum-starved cells (p < 0.05). In comparison with control cells, rapamycin-treated cells exhibited reduced proliferation, similar to serum-starved cells. The viability (as assessed by the MTT assay) of D3-1R-treated cells was good, similar to control cells, showing their quality was maintained. To confirm nutrient regulation by rapamycin treatment, we checked the transcript levels of nutrient transporter genes (SLC2A2, SLC2A4, SLC6A14, and SLC7A1). These levels were significantly lower in D3-1R-treated cells than in control cells (p < 0.01). We performed SCNT with D3-1R-treated cells (SCNT(D3-1R)) to confirm the effect of cell cycle synchronization by rapamycin treatment. Although SCNT(D3-1R) embryos did not have an increased fusion rate, their cleavage and blastocyst formation rates were significantly higher than those of control embryos (p < 0.05). Regarding embryo quality, the numbers of total and apoptotic cells per blastocyst were increased and decreased, respectively, in SCNT(D3-1R) blastocysts. The mRNA levels of developmental (CDX2 and CDH1) and proapoptotic (FAS and CASP3) genes were significantly higher and lower, respectively, in SCNT(D3-1R) blastocysts than in control blastocysts (p < 0.05). These results demonstrate that rapamycin treatment affects the cell cycle synchronization of donor cells and enhances the developmental potential of porcine SCNT embryos.

Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.