Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells' proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.
Background
Centrally located cytoplasmic granulation (CLCG) is a common cytoplasmic dysmorphism in human oocytes retrieved after controlled ovarian hyperstimulation (COH). This study sought to achieve a better understanding of its formation and effects on clinical outcomes.
Methods
We retrospectively analyzed the data from 422 intracytoplasmic sperm injection (ICSI) treatment cycles. Three groups of patients were classified according to the rates of CLCG occurrence in one egg cohort, as partial (pCLCG) all (aCLCG) and no CLCG (control).
Results
The pCLCG group had a significantly lower Body Mass Index (BMI) and higher Anti-Mullerian hormone (AMH) level compared to the control or aCLCG groups. Consistent with these distinctive features in the pCLCG group, fertilization and blastocyst formation rates were reduced significantly in the pCLCG group but not in the aCLCG group. Furthermore, the clinical outcomes in fresh embryo transfer cycles were dramatically reduced in the pCLCG group compared with the control group. However, in frozen/thawed cycles, all three clinical outcomes were significantly reduced in the aCLCG group but not in the pCLCG group.
Conclusion
We propose that pCLCG may reflect a specific population of patients, and that the CLCG structure is sensitive to cryopreservation.
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