A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become an essential research platform to study different human diseases once being discovered by Dr. Shinya Yamanaka in 2006. Another breakthrough in biomedical research is the application of CRISPR/Cas9 system for genome editing in mammalian cells. Although numerous studies have been done to develop methods for gene editing in iPSCs, the current approaches suffer from several limitations, including time and labor consuming, low editing efficiency, and potential off-target effects. In the current study, we report an electroporation-mediated plasmid CRISPR/Cas9 delivery approach for genome editing in iPSCs. With this approach, an edited iPSC cell line could be obtained within 2 weeks. In addition, the transit introducing of CRISPR/Cas9 machinery could minimize genomic integration of Cas9 gene, which avoided potential long-term side effects of Cas9 enzyme. We showed that CRISPR/Cas9-mediated genomic editing did not affect pluripotency and differentiation ability of iPSCs. With the quickly evolving of both iPSC and CRISPR/Cas9-mediated genome editing research fields, we believe that our method can significantly facilitate the application of genome editing in iPSCs research.

Diabetes inhibits corneal epithelial cell migration and tight junction formation in mice and human via increasing ROS and impairing Akt signaling.

Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 µM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.