AMC and RC are co-inventors on US Patent no. 9,198,911 filed by Columbia University and licensed to Aclaris Therapeutics. RC is a consultant for and shareholder of Aclaris Therapeutics. AMC is a consultant for Aclaris Therapeutics and Dermira and is a shareholder of Aclaris Therapeutics. AMC has received grant support from Pfizer.
The primary hepatocytes were extracted and purified from mice through improved Seglen two-step perfusion method. Ethanol-induced injury hepatocytes model in mice was used to investigate the importance of glutathione S-transferase A1 (GSTA1) in hepatocytes injury by comparison with other indicators, such as alanine aminotransferase, aspartate aminotransferase, malondialdehyde, glutathione and superoxide dismutase. The release of GSTA1 was demonstrated to be an earlier and more sensitive indicator of hepatocytes injury than other indicators. Significant increases in GSTA1 were detected at 2 h after ethanol exposure, while other indicators were undetected at this time. A markedly difference in other indicators were observed at 6 and 8 h. The release of GSTA1 was significantly increased at a concentration of 50 mmol/L ethanol, the lowest exposure concentration than that in other indicators. In contrast, other indicators release was not statistically significant until concentrations of 75 mmol/L and 100 mmol/L ethanol. These results suggest that GSTA1 can be detected at the early stage of low concentration ethanol exposure and that GSTA1 is more sensitive and reliable marker in ethanol-induced hepatic injury.
Pluripotent stem cells (PSCs) generated from somatic cells via ectopic expression of specific transcription factors provide an unlimited cell resource for regenerative medicine and transgenic breeding. Here, we describe the successful generation of bovine induced PSCs (biPSCs) from foetal fibroblasts by lentivirus-mediated delivery of bovine pluripotency reprogramming factors (PRFs) OCT3/4, SOX2, KLF4, c-MYC, NANOG and LIN28. The generated biPSCs resembled embryonic stem cells (ESCs) in their gene expression profiles, self-renewal capabilities and proliferation, as well as maintenance of a normal karyotype and differentiation into diverse cell types of all three germ layers both in vitro and in vivo. Qualitative phosphoproteomics of biPSCs revealed a large number of phosphorylated proteins, which might be related to the control of biPSCs status. The successful generation of biPSCs and the analysis of their phosphoproteome would further our understanding of the epigenetic mechanisms underlying iPSC pluripotency, thus promoting their application in bovine transgenic breeding and marking avenues for future research.
The consistent failure to isolate bona fide pluripotent cell lines from livestock indicates that the underlying mechanisms of early lineage specification are poorly defined. Unlike other species, the contrivances of segregation have been comprehensively studied in the mouse. In mouse, FGF/MAPK signalling pathway dictates the segregation of hypoblast (primitive endoderm). However, it is not evident whether this mechanism is also conserved in livestock. Here, in this study, we examined the roles of FGF/MAP kinase signalling pathways in porcine parthenogenetic embryos during the early development. Porcine parthenogenetic embryos were cultured in the medium addition with FGFR inhibitor BGJ398 (10 μm) or DEMOS. Pluripotency- and lineage-related gene expressions in the early porcine embryos were determined. Compared to control, total cell numbers on day 7 were significantly higher (55 ± 5.96 vs 47 ± 1.97, p < 0.05) in embryos cultured in the presence of BGJ398, but had no significant effect on the rate of blastocyst development (47% vs 44%, p > 0.05). Nonetheless, BGJ398 treatment significantly augmented the expression of pluripotency and trophoblast marker genes (SOX2, OCT4, KLF4 and CDX2), but did not significantly change the expression of NANOG and hypoblast marker gene (GATA4). Furthermore, the addition of FGF signalling agonist (FGF2) during the embryo development significantly decreased the expression of pluripotency and trophoblast marker genes (SOX2, NANOG, KLF4 and CDX2), but no significant effect on the expression of OCT4 and GATA4 was observed. Here, we exhibit that inhibition of FGF signalling could improve the quality of the porcine embryo and escalate the chance to capture pluripotency. Besides, it also promotes the trophoblast development of porcine parthenogenetic embryo. In addition, the data suggested that FGF signalling pathway is dispensable for the segregation of hypoblast and epiblast lineages in porcine embryo during the early development.
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