Differential susceptibility to lipopolysaccharide affects the activation of toll-like-receptor 4 signaling in THP-1 cells and PMA-differentiated THP-1 cells.

Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines-IL-1ß, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.

Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII.p12_GII.3 Human Norovirus by mRNA Sequencing.

Norovirus genogroup II (NoV GII) induces acute gastrointestinal food-borne illness in humans. Because gnotobiotic pigs can be infected with human norovirus (HuNoV) GII, they are frequently used to analyze the associated pathogenic mechanisms and immune responses, which remain poorly understood. Recently, mRNA sequencing analysis (RNA-Seq) has been used to identify cellular responses to viruses. In this study, we investigated the host immune response and possible mechanisms involved in virus evasion in the ileum of gnotobiotic pigs infected with HuNoV by RNA-Seq. HuNoV was detected in the feces, blood, and tissues of the jejunum, ileum, colon, mesenteric lymph node, and spleen of pigs infected with HuNoV. In analysis of mRNA sequencing, expression of anti-viral protein genes such as OAS1, MX1, and MX2 were largely decreased, whereas type I IFN was increased in pigs infected with HuNoV. In addition, expression of TNF and associated anti-inflammatory cytokine genes such as IL10 was increased in HuNoV-infected pigs. Expression of genes related to natural killer (NK) cell cytotoxicity and CD8+ T cell exhaustion was increased, whereas that of MHC class I genes was decreased. Expression profiles of selected genes were further confirmed by qRT-PCR and Western blot. These results suggest that infection with HuNoV induces NK cell-mediated cytotoxicity but suppresses type I IFN- and CD8+ T cell-mediated antiviral responses.

Comparative analysis of immune related genes between domestic pig and germ-free minipig.

Recently, minipig has been considered as an animal model that is appropriate for human disease model to study toxicology, pharmacology, and xenotransplantation. Nevertheless, minipigs are bred in various environment according to their use. Here, we suggest that minipigs used for research should be bred in well-controlled facility, comparing immune status of pigs raised in different breeding environment. DNA microarray was performed with ear skin and placenta of Landrace domestic pigs (DPs) and Minnesota germ-free minipigs (GPs). Their immune transcriptome was analyzed by gene ontology (GO) annotation database, based on criteria of |log2 fold change| ≥1 with P ≤ 0.05. As a result, we found that immune related genes in the ear skin of DPs were highly activated, compared to GPs. On the other hand, no significant s were found in the placenta. Quantitative real-time PCR (qRT-PCR) was performed in five candidate immune genes. Their fold changes were consistent with the results from DNA microarray (P ≤ 0.05). In conclusion, we experimentally proved that porcine immune system was affected by different breeding environment, suggesting the importance of controlling microbes in animal room for the qualified research.

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization.

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.

Association Between Functional Activity of Mitochondria and Actin Cytoskeleton Instability in Oocytes from Advanced Age Mice.

Mitochondrial dysfunction is strongly associated with the oocyte quality and aging, wherein the aged oocytes are related to the actin cytoskeleton integrity; however, whether this integrity is associated with mitochondrial dysfunction in oocytes from aged mice remains unclear. In the present study, we investigated the relationship between mitochondrial dysfunction and actin cytoskeleton instability in oocytes from the aged mice. We performed comparable analysis of mitochondrial motility between young, 1.5 µM cytochalasin B (CB)-treated young oocytes, and aged oocytes by confocal live imaging. Moreover, we analyzed the relationships between mitochondrial motility and maturation ratios, including ATP production ratio of the young, CB-treated young, and aged oocytes. Actin cytoskeleton instability in the aged oocytes and CB-treated young oocytes led to a significant decrease in the mitochondrial motility and low ATP productive ratios compared to those in the young group. Our data suggest that the actin cytoskeleton instability is presumably the primary cause for the loss of mitochondrial function in the aged murine oocytes.

Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF.

In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.

5-Azacytidine improves the meiotic maturation and subsequent in vitro development of pig oocytes.

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2'-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0-10 µM). Cloned embryos generated from 5-Aza-C (0.01 µM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ±â€¯1.5% compared with 9.6 ±â€¯1.8%), greater probability of hatching (61.8 ±â€¯1.5% compared with 45.0 ±â€¯3.9%) and contained a greater number of cells per blastocyst (38.5 ±â€¯4.4 compared with 30.5 ±â€¯3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research.

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24 h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3 K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2 h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.

The critical warning sign of real-time brainstem auditory evoked potentials during microvascular decompression for hemifacial spasm.

OBJECTIVE: The aim of this study was to define the critical warning sign of real-time brainstem auditory evoked potential (BAEP) for predicting hearing loss (HL) after microvascular decompression (MVD) for hemifacial spasm (HFS). METHODS: Nine hundred and thirty-two patients with HFS who underwent MVD with intraoperative monitoring (IOM) of BAEP were analyzed. We used a 43.9 Hz/s stimulation rate and 400 averaging trials to obtain BAEP. To evaluate HL, pure-tone audiometry and speech discrimination scoring were performed before and one week after surgery. We analyzed the incidence for postoperative HL according to BAEP changes and calculated the diagnostic accuracy of significant warning criteria. RESULTS: Only 11 (1.2%) patients experienced postoperative HL. The group showing permanent loss of wave V showed the largest percentage of postoperative HL (p < 0.001). No patient who experienced only latency prolongation (≥1 ms) had postoperative HL. Loss of wave V and latency prolongation (≥1 ms) with amplitude decrement (≥50%) were highly associated with postoperative HL. CONCLUSIONS: Loss of wave V and latency prolongation of 1 ms with amplitude decrement ≥50% were the critical warning signs of BAEP for predicting postoperative HL. SIGNIFICANCE: These findings elucidate the critical warning sign of real-time BAEP.

Sirtuin inhibition leads to autophagy and apoptosis in porcine preimplantation blastocysts.

Sirtuins are nicotinamide adenine dinucleotide dependent class III histone deacetylase proteins that play a crucial role in several cellular processes, including DNA repair, apoptosis, and lifespan. Previous studies have shown that sirtuin inhibition leads to embryonic developmental arrest and oxidative stress in porcine and murine. However, sirtuin-mediated mechanisms have not been examined in porcine preimplantation blastocysts. We therefore investigated the relationship between sirtuins and autophagy. Embryos were cultured with 100 µM sirtinol (SIRT1/2 inhibitor) in NCSU-23 media after in vitro fertilization. Treatment with sirtinol significantly reduced the rates of morula (21.34 ± 1.84 vs. 11.89 ± 2.01), blastocyst development (17.18 ± 1.81 vs. 9.00 ± 2.02), and total cell number (50.80 ± 1.47 vs. 37.71 ± 1.79), compared to controls, with an associating decrease the levels of Sirt2 transcript. Sirtinol treatment induced autophagy through an increase in LC3 transcript and LC3 protein. BECLIN1 and ATG5 expression showed a slight increase in treated group. Finally, treatment with sirtinol dramatically increased TUNEL indices (6.55 ± 0.84 vs. 11.44 ± 0.81) and fragmentation indices (0.33 ± 0.05 vs. 1.40 ± 0.30). BCL2L1 expression was lower, while Caspase-3 expression was significantly elevated in the sirtinol-treated group. Therefore, these findings suggest that sirtuins may elicit their effects through modifying autophagy and apoptosis, leading to developmental arrest and reducing the quality of porcine preimplantation embryos.

Human galectin-9 on the porcine cells affects the cytotoxic activity of M1-differentiated THP-1 cells through inducing a shift in M2-differentiated THP-1 cells.

BACKGROUND: In xenotransplantation, immune rejection by macrophages occurs rapidly and remains a major obstacle. Studies to control immune rejection in macrophages have been continuing to date. Recent studies have reported that human galectin-9 (hGal-9) can regulate the function of regulatory T cells (Treg), as well as cytotoxicity T cells (CTL) and natural killer cells (NK). Although the effect of hGal-9 on lymphocytes has been well studied, the relationship between hGal-9 and myeloid cells has been scarcely studied. METHODS: To confirm the decreased cytotoxic activity effect by hGal-9 in M1-differentiated THP-1 cells, we established the hGal-9 expressing transgenic porcine cell line. hGal-9 siRNA was transfected to transgenic cells and recombinant hGal-9 (rhGal-9) was treated to co-culturing condition, and then, flow cytometry assay was conducted for analyzing the cytotoxic activity of M1-differentiated THP-1 cells. Related inflammatory cytokines (IL-1ß, IL-10, TNF-α, IL-6, IL-12, IL-23, and TGF-ß) and related enzymes (iNOS and Arginase 1) were analyzed by qPCR and Western blot assay. To identify the shift in M1/M2-differentiated THP-1 cells, expression levels of CCR7, CD163, iNOS, and Arginase 1 and population of M2 marker positive cells were analyzed. RESULTS: The expression levels of pro-inflammatory cytokines in M1-differentiated THP-1 cells co-cultured with hGal-9-expressing porcine kidney epithelial cells were decreased, but not in co-cultured THP-1 cells. However, the expression levels of anti-inflammatory cytokines were also increased in co-cultured M1-differentiated THP-1 cells. The cytotoxicity effect of M1-differentiated THP-1 cells on transgenic cells was decreased while the expression levels of anti-inflammatory cytokines and M2 macrophages-related molecules were increased. M2 differentiation program was turned on while M1 program was turned down by enhancing the phosphorylation levels of Akt and PI3K and the expression level of PPAR-γ. Due to these changes, differentiation of M2 program was enhanced in cells co-cultured with hGal-9. CONCLUSIONS: These data suggested that hGal-9 has a reduction in M1-differentiated THP-1 cell cytotoxic activity-related acute immune rejection in pig-to-human xenotransplantation in addition to its role in lymphoid lineage immune cell regulation.

The potentiating effect of hTFPI in the presence of hCD47 reduces the cytotoxicity of human macrophages.

BACKGROUND: In pig-to-human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3-galactosyltransferase knockout pigs. However, macrophage-mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD47 inhibits monocyte/macrophage-mediated phagocytosis by identifying the CD47 signal regulatory protein alpha (SIRP-α) as self/non-self. Tissue factor pathway inhibitor (TFPI) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD47, thrombospondin-1 (TSP-1). When TSP-1 binds to CD47, phagocytosis in macrophages is increased. METHODS: The 2A peptide system was used to establish pig kidney cells (PK15) simultaneously expressing human CD47 and human TFPI, and they were cultured with activated THP-1 cells. After staining with 7-aminoactinomycin D, flow cytometry analysis was carried out. TFPI siRNA analysis and recombinant human TFPI (rhTFPI) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP-1 cells in the presence of CD47. Related inflammatory cytokines produced by activated THP-1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was analyzed using immunoprecipitation and Western blot. RESULTS: hCD47/hTFPI-PK15 cells survived better than hCD47-PK15, hTFPI-PK15, or normal PK15 cells on cytotoxicity tests using activated THP-1 cells. TSP-1, derived from these activated THP-1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells (PBMCs). The tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was further increased in the case of co-expression of CD47/TFPI than in individual non-expression or expression of CD47 or TFPI alone. CONCLUSIONS: When hCD47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP-α in activated THP-1 cells via hTSP-1 inhibition, and consequently, it might improve the effect of hCD47-SIRP-a signaling.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation.

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

Neogenin regulates mitochondrial activity in pre-implantation mouse embryos.

• Mitochondrial activation signaling pathways are not clearly identified. • Embryonic mitochondria activity is important for a successful pregnancy and live birth. • Neogenin is a multi-functional receptor that contributes to embryo development. • Neogenin as a receptor is related to mitochondrial activation and replication. • Neogenin could activate mitochondria in pre-implantation embryo development.

Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts.

The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.

The regulation of autophagy in porcine blastocysts: Regulation of PARylation-mediated autophagy via mammalian target of rapamycin complex 1 (mTORC1) signaling.

Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6K-thr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos.

Induced neural stem cells from distinct genetic backgrounds exhibit different reprogramming status.

Somatic cells could be directly converted into induced neural stem cells (iNSCs) by ectopic expression of defined transcription factors. However, the underlying mechanism of direct lineage transition into iNSCs is largely unknown. In this study, we examined the effect of genetic background on the direct conversion process into an iNSC state. The iNSCs from two different mouse strains exhibited the distinct efficiency of lineage conversion as well as clonal expansion. Furthermore, the expression levels of endogenous NSC markers, silencing of transgenes, and in vitro differentiation potential were also different between iNSC lines from different strains. Therefore, our data suggest that the genetic background of starting cells influences the conversion efficiency as well as reprogramming status of directly converted iNSCs.

Poly(ADP-ribosyl)ation is involved in pro-survival autophagy in porcine blastocysts.

Poly(ADP-ribosyl)ation (PARylation) prevents apoptosis through its involvement in pro-survival autophagy in cultured cells; whether or not the same is true for pre-implantation embryos has not yet been documented. In this study, we investigated the participation of PARylation and autophagy in in vitro porcine pre-implantation embryo development. The transcript levels of autophagy-related genes and poly(ADP-ribose) polymerase 1 (PARP1), an enzyme required for PARylation, were transiently up-regulated by fertilization, decreased at the late 1-cell stage, and maintained until the blastocyst stage. LC3, a marker of autophagosomes, and poly(ADP-ribose) (PAR) polymer were present in all stages of pre-implantation development. Exposure of embryos to 3-methyladenine, an autophagy inhibitor, or 3-aminobenzamide, a PARP inhibitor, suppressed the development of blastocysts. Pharmacological inhibition of PARylation further suppressed pro-survival autophagy by decreasing the expression of autophagy-related genes (ATG5, BECLIN1, and LC3) and decreasing LC3 protein abundance while increasing the rate of apoptosis in blastocysts. Deficiency in autophagy also induced abnormal accumulation of SQSTM1/p62 aggregates in porcine blastocysts. Collectively, these data suggest that PARylation is involved in selective autophagic degradation of ubiquitinated proteins, functioning in a pro-survival role, in porcine in vitro-produced embryos. These pro-survival regulatory mechanisms may be important for the control of embryo quality.