Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States.

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFß signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic ß-Cell Line.

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic ß-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic ß-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic ß-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC-/-) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC-/- MIN6 cells. Therefore, α-ENaC-/- MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic ß-cells.

Clinical Significance of 4L Lymph Node Dissection in Left Lung Cancer.

PURPOSE: To investigate the prognostic impact of 4L lymph node (LN) dissection in left lung cancer and to analyze the relative risk factors for 4L LN metastasis. PATIENTS AND METHODS: We retrospectively collected data from 657 patients with primary left lung cancer who underwent surgical pulmonary resection from January 2005 to December 2009. One hundred thirty-nine patients underwent 4L LN dissection (4LD+ group); the other 518 patients did not receive 4L LN dissection (4LD- group). Propensity score weighting was applied to reduce the effects of observed confounding between the two groups. Study end points were disease-free survival (DFS) and overall survival (OS). RESULTS: The metastasis rate of station 4L was 20.9%, which was significantly higher than those of station 7 (14.0%; P = .048) and station 9 (9.8%; P < .001). Station 4L metastasis was associated with most other LN station metastases in univariate analysis, but only station 10 LN metastasis was an independent risk factor for 4L LN metastasis (odds ratio, 0.253; 95% CI, 0.109 to 0.588; P = .001) in multivariate logistic analysis. The 4LD+ group had a significantly better survival than the 4LD- group (5-year DFS, 54.8% v 42.7%; P = .0376; 5-year OS, 58.9% v 47.2%; P = .0200). After allowing potential confounders in multivariate survival analysis, dissection of 4L LN retained its independent favorable effect on DFS (hazard ratio, 1.502; 95% CI, 1.159 to 1.947; P = .002) and OS (hazard ratio, 1.585; 95% CI, 1.222 to 2.057; P = .001). Propensity score weighting further confirmed that the 4LD+ group had a more favorable DFS ( P = .0014) and OS ( P < .001) than the 4LD- group. CONCLUSION: Station 4L LN involvement is not rare in left lung cancer, and dissection of the 4L LN station seems to be associated with a more favorable prognosis as compared with those who did not undergo this dissection.