A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.

m6A demethylase ALKBH5 suppression contributes to esophageal squamous cell carcinoma progression.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant gastrointestinal cancer with a high recurrence rate and poor prognosis. Although N6-methyladenosine (m6A), the most abundant epitranscriptomic modification of mRNAs, has been implicated in several cancers, little is known about its participation in ESCC progression. We found reduced expression of ALKBH5, an m6A demethylase, in ESCC tissue specimens with a more pronounced effect in T3-T4, N1-N3, clinical stages III-IV, and histological grade III tumors, suggesting its involvement in advanced stages of ESCC. Exogenous expression of ALKBH5 inhibited the in vitro proliferation of ESCC cells, whereas depletion of endogenous ALKBH5 markedly enhanced ESCC cell proliferation in vitro. This suggests ALKBH5 exerts anti-proliferative effects on ESCC growth. Furthermore, ALKBH5 overexpression suppressed tumor growth of Eca-109 cells in nude mice; conversely, depletion of endogenous ALKBH5 accelerated tumor growth of TE-13 cells in vivo. The growth-inhibitory effects of ALKBH5 overexpression are partly attributed to a G1-phase arrest. In addition, ALKBH5 overexpression reduced the in vitro migration and invasion of ESCC cells. Altogether, our findings demonstrate that the loss of ALKBH5 expression contributes to ESCC malignancy.

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

The diagnostic value of [18F]-FDG-PET/CT in assessment of radiation renal injury in Tibet minipigs model.

BACKGROUND: Radiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys. METHODS: Forty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples' and renal function analysis. RESULTS: The uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy. CONCLUSIONS: 18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.

Effect of Prepregnancy Obesity on Litter Size in Primiparous Minipigs.

Obesity is a public health problem in both developed and developing countries, and the negative effects of obesity on reproductive physiology have been highlighted recently. We evaluated the effects of porcine obesity index, sex hormones, and peptide hormones on litter size in various breeds of minipigs. Blood samples were collected from sedated 8-,10-, and 12-mo-old minipigs to measure preovulatory levels of sex hormones (follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, testosterone, and prolactin) and peptide hormones (insulin-like growth factor, glucagon, cortisol, growth hormone, free thyroxine, free triiodothyronine, insulin, and leptin). We also measured weight, abdominal circumference, neck circumference, and body length and then calculated the porcine obesity index. Data were analyzed by one-way ANOVA, and means were compared by least significance difference testing. Pearson correlation between parameters and litter size was analyzed. Prepregnancy porcine obesity index and litter size were negatively correlated in primiparous minipigs. Litter size was influenced by luteinizing hormone, estradiol, progesterone, testosterone, prolactin, follicle-stimulating hormone, cortisol, insulin-like growth factor 1, growth hormone, free thyroxine, insulin, and leptin. In conclusion, prepregnancy obesity reduces litter size in primiparous minipigs.

Poly[(µ(4)-biphenyl-2,4'-dicarboxyl-ato-κ(5)O(2):O(2'):O(4):O(4),O(4'))zinc].

The crystal structure of the polymeric title complex, [Zn(C(14)H(8)O(4))](n), is composed of layers parallel to (110) formed by linking of Zn-carboxyl-ate chains with biphenyl units of the biphenyl-2,4'-dicarboxyl-ate (bpdc) ligands. The Zn(II) atom is five-coordinated in a distorted square-pyramidal geometry by five O atoms from four bpdc ligands. The dihedral angle between the benzene rings is 52.32 (12)°.