Fungal Community Analyses of Endophytic Fungi from Two Oak Species, Quercus mongolica and Quercus serrata, in Korea.

Fungal endophytes have been recorded in various plant species with a richness of diversity, and their presence plays an essential role in host plant protection against biotic and abiotic stresses. This study applied the Illumina MiSeq sequencing platform based on the amplification of fungal ribosomal ITS2 region to analyze fungal endophytic communities of two oak species (Quercus mongolica and Q. serrata) with different oak wilt disease susceptibilities in Korea. The results showed a total of 230,768 sequencing reads were obtained and clustered at a 97% similarity threshold into 709 operational taxonomic units (OTUs). The OTUs of Q. serrata were higher than that of Q. mongolica with the number of 617 OTUs and 512 OTUs, respectively. Shannon index also showed that Q. serrata had a significantly higher level of fungal diversity than Q. mongolica. Total of OTUs were assigned into 5 fungal phyla, 17 classes, 60 orders, 133 families, 195 genera, and 280 species. Ascomycota was the dominant phylum with 75.11% relative abundance, followed by Basidiomycota with 5.28%. Leptosillia, Aureobasidium and Acanthostigma were the most abundant genera detected in Q. serrata with the average relative abundance of 2.85, 2.76, and 2.19%, respectively. On the other hand, Peltaster, Cladosporium and Monochaetia were the most common genera detected in Q. mongolica with the average relative abundance of 4.83, 3.03, and 2.87%, respectively. Our results indicated that fungal endophytic communities were significantly different between two oak species and these differences could influence responses of host trees to oak wilt disease caused by Raffaelea quercus-mongolicae.

The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells.

Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.

Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased (p < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.