Derivation of Putative Porcine Embryonic Germ Cells and Analysis of Their Multi-Lineage Differentiation Potential

Embryonic germ （EG） cells are cultured pluripotent stem cells derived from the primordial germ cells （PGCs） that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22-30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24-28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase （AP）. These cells were able to form embryoid bodies （EBs） in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein （AFP）, s-smooth muscle actin （^-SMA）, and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunoftuorescence for neuronal class III [3-tubulin （Tuj 1）, glial fibrillary protein （GFAP） and galactosylceramidase （GALC）, respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.

On-chip multiplexed single-cell patterning and controllable intracellular delivery

Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo.These limitations include low efficiency,noticeable cell damage and nonuniform delivery of cells.Here,we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules.A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method.A controllable vacuum system was adopted to trap a single cell on each micropore.Using this chip,safe single-cell electroporation was performed at low voltage.Cargoes of various sizes ranging from oligonucleotides(molecular beacons,22 bp)to plasmid DNA(CRISPR-Cas9 expression vectors,>9 kb)were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods(e.g.,commercial electroporation devices and Lipofectamine).The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage.By using CRISPR-Cas9 transfection with this system,the p62 gene and CXCR7 gene were knocked out in tumor cells,which effectively inhibited their cellular activity.Overall,this vacuum-assisted micropore array platform provides a simple,efficient,high-throughput intracellular delivery method that may facilitate on-chip cell manipulation,intracellular investigation and cancer therapy.

Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

The aim of this study was to optimize electrofusion conditions for generating porcine tet- raploid （4n） embryos and produce tetraploid/diploid （4n/2n） chimeric embryos. Different electric field intensities were tested and 2 direct current （DC） pulses of 0.9 kV/em for 30 p.s was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization （FISH）. Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts （P 〈 0.05）, there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos （P 〉 0.05）, suggesting that the blas- tocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell （4n）/4-cell （2n） chimeric embryos were significantly higher than those of 2-cell （4n）/4-cell （2n）, 4-cell （4n）/8-cell （2n）, 4-cell （4n）/2-cell （2n） chimeric embryos （P 〈 0.05）. Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm （TE）, while the 2n cells are mainly present in the inner cell mass （ICM） of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

Targeting cytokinesis bridge proteins to kill high-CIN type tumors

As a common feature of tumors,chromosomal instability(CIN)not only forces carcinomatous evolution,but also loads cancer cells with extra pressure through a robust imbalance of genome patterning that may be used for cancer treatment.Errors in cytokinesis increase CIN,so cytokinesis components are valuable targets for treating cancer.However,due to the short time span and confined space of cytokinesis bridges,profiling cytokinesis fac-tors is challenging.Taking advantage of engineered ascorbate peroxidase(APEX2),we established a cytokinesis bridge-APEX reaction in living cells.A total of 218 cytokinesis bridge proteins were identified with high relia-bility.Knockdown of cytokinesis bridge genes generated micronuclei that activate the cGAS-pathway and cause apoptosis in cancer cells bearing high CIN rather than low CIN.Thus,our study proposes a strategy for killing high-CIN tumors regardless of tumor type,and provides a proteome resource of cytokinetic bridges for future research.