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Past researches have shown that pluripotency maintenance of naive and primed-state pluripotent stem cells (PSCs) depends on different signaling pathways, and naive-state PSCs possess the ability to produce chimeras when they are introduced into a blastocyst. Considering porcine is an attractive model for preclinical studies, many researches about pig induced pluripotent stem cells (piPSCs) have been reported. Some cytokines and small molecule compounds could transform primed piPSCs into naive state. However, there are no suitable culture conditions for generation of naive-state piPSCs with high efficiency; other small molecule compounds need further exploration. In this study, we investigated whether p38 MAPK and JNK signal pathway inhibitor SB203580 and SP600125 could be of benefit for acquiring naive-state piPSCs. By comparing reprogramming efficiencies under conditions of different donor cells and culture environment, we found that porcine bone marrow mesenchymal stem cells (PBMSCs) have higher efficiency on piPSC induction, and the culture condition of CHIR99021+PD0325901(2i)+Lif+bFGF is more suitable for subculturing of piPSCs. Our results also indicate that SB203580 and SP600125 could promote reprogramming of PBMSCs into naive-like state piPSCs. These results provide guidance for choosing donor cells, culture conditions, and research of different state iPSCs during the process of reprogramming pig somatic cells.

Signal transducer and activator 5 (STAT5) plays important roles in regulating mammary glandular cell proliferation and milk-protein gene expression. However, the functions of STAT5a and STAT5b genes in lactation of buffalo remain uninvestigated. In this study, full-length STAT5a (2502 bp) and STAT5b (2515 bp) coding sequences were isolated for the first time. The highest STAT5a gene expression was found in buffalo mammary glands and the highest STAT5b gene expression was found in buffalo brains and mammary glands. HE staining indicated that STAT5a and STAT5b are mainly expressed in epithelial cells of buffalo mammary glands. Next, we investigated the functions of STAT5 by knocking down and overexpressing STAT5 in buffalo mammary epithelial cells (BuMECs). According to our results, STAT5 knockdown resulted in inhibited G1/S transition of BuMECs and significantly lower expression of milk-protein genes, whereas overexpression of STAT5 resulted in significantly higher expression of milk-protein genes. In summary, our results demonstrate that STAT5 can regulate the cell cycle transition of BuMECs and affect the expression of milk-protein genes. Our research lays a foundation for further study of the role of

Pig-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. The miR-302s cluster alone has been shown to reprogram mouse and human somatic cells into induced pluripotent stem cells (iPSCs) without exogenous transcription factors. However, miR-302s alone have not been reported to reprogram cells in large livestock. In this study, we induced pig somatic cells into partially reprogrammed piPSCs using overexpression of the miR-302s cluster (miR-302s-piPSC) and investigated the early reprogramming events during the miRNA induction process. The results showed that miR-302s-piPSCs exhibited some characteristics of pluripotent stem cells including expression of pluripotency markers-particularly, efficient activation of endogenous OCT4-and differentiation to the three germ layers in vitro. During the early reprogramming process, somatic cells first underwent epithelial-mesenchymal transition and then mesenchymal-epithelial transition to eventually form miR-302s-piPSCs. These data show, for the first time, that single factor miR-302s successfully induced pig somatic cells into miR-302s-piPSCs. This study provides a new tool and research direction for the induction of pluripotent stem cells in a large livestock.

CD14 plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, resulting in inflammation. Upstream inhibition of the inflammation pathway mediated by bacterial LPS, toll-like receptor 4 (TLR4) and cluster of differentiation antigen 14 (CD14) was proven to be an effective therapeutic approach for attenuating harmful immune activation. To explore the effect of CD14 downregulation on the expression of TLR4 signaling pathway-related genes after LPS stimulation in buffalo (Bubalus bubalis) monocyte/macrophages, effective CD14 shRNA sequences were screened using qRT-PCR and FACS analysis with buffalo CD14 shRNA lentiviral recombinant plasmids (pSicoRGFP-shRNA) and buffalo CD14 fusion expression plasmids (pDsRed-N1-buffalo CD14) co-transfected into HEK293T cells via liposomes. Of the tested shRNAs, shRNA-1041 revealed the highest knockdown efficiency (p < 0.01). When buffalo peripheral blood monocyte/macrophages were infected with shRNA-1041 lentivirus and stimulated with LPS, the expression of endogenous CD14 was significantly decreased by CD14 shRNA (p < 0.01), and the mRNA expression levels of TLR4, IL-6 and TNF-α were also significantly downregulated compared to the control groups (p < 0.01). These results demonstrated that the knockdown of endogenous CD14 had clear regulatory effects on the signal transduction of TLR4 after stimulation with LPS. These results may provide a better understanding of the molecular mechanisms of CD14 regulation in the development of several buffalo diseases.

Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock. This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).