Tai Hyun Park

Regulation of morphogenesis and neural differentiation of human mesenchymal stem cells using carbon nanotube sheets

In order to successfully utilize stem cells for therapeutic applications in regenerative medicine, efficient differentiation into a specific cell lineage and guidance of axons in a desired direction is crucial. Here, we used aligned multi-walled carbon nanotube (MWCNT) sheets to differentiate human mesenchymal stem cells (hMSCs) into neural cells. Human MSCs present a preferential adhesion to aligned CNT sheets with longitudinal stretch parallel to the CNT orientation direction. Cell elongation was 2-fold higher than the control and most of the cells were aligned on CNT sheets within 5° from the CNT orientation direction. Furthermore, a significant, synergistic enhancement of neural differentiation was observed in hMSCs cultured on the CNT sheets. Axon outgrowth was also controlled using nanoscale patterning of CNTs. This CNT sheet provides a new cellular scaffold platform that can regulate morphogenesis and differentiation of stem cells, which could open up a new approach for tissue and stem cell regeneration.

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14xa0%, respectively), while the control cells produced predominantly bi-antennary branching (70xa0%). About 53xa0% of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77xa0%, whereas only 61xa0% of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6xa0% after the addition of 0.2xa0mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

A protein delivery system using 30Kc19 cell-penetrating protein originating from silkworm

Cell-penetrating protein and its protein transduction domain have been used to deliver drugs and proteins into the cells via receptor-independent endocytosis. A number of cell-penetrating proteins including TAT derived from HIV-1 virus, VP22 from herpes simplex virus and Antennapedia from drosophila have been discovered. Here, we report a cell-penetrating protein, 30Kc19, originating from the hemolymph of silkworm, Bombyx mori. The 30Kc19 is the first cell-penetrating protein that has been found in insect hemolymph. When the 30Kc19 protein produced from recombinant Escherichia coli was supplemented into the medium for mammalian cell culture, 30Kc19 efficiently penetrated into various types of cells and localized at subcellular compartments including mitochondria and cytoplasm. 30Kc19 also delivered cargo proteins such as green fluorescence protein into the cells even though cargo proteins are not able to penetrate into cells by themselves. In addition to the inxa0vitro system, 30Kc19 exhibited the protein transduction property inxa0vivo. When 30Kc19 was intraperitoneally injected into mice, 30Kc19 delivered cargo proteins into various organ tissues of model animals without producing toxicity. Therefore, 30Kc19 has a great potential as a cell-penetrating protein that can be used as a medicinal tool to deliver cargo molecules including proteins into the target organ tissues in the body.

Antioxidant effect of protein-free silkworm hemolymph extract in mitochondrial membrane potential

Silkworm was used in the sericulture industry and also processed for food sources in some of Asian countries. In previous studies, it was reported that the 30K protein in silkworm, Bombyx mori hemolymph inhibited animal cell apoptosis. In this work, the antioxidative function of silkworm hemolymph was investigated using its protein-free extract. H2O2-induced ROS and O2·− were efficiently scavenged in Chinese hamster ovary cells when the culture medium was supplemented with silkworm hemolymph. The hemolymph extract prepared by deproteinization through ethanol precipitation completely scavenged DPPH radicals (IC50=0.025 mg/mL). When the extract was supplemented with protein-free medium, oxidative damage to mitochondria was efficiently diminished with the maintenance of the mitochondrial membrane potential. Lutein was identified as the major antioxidative constituents in the extract (52.06 μg/mg).

The cultivation of Anabaena variabilis in a bubble column operating under bubbly and slug flows.

In a bubble column reactor with an inner diameter of 6cm and a height of 63cm for the culture of cyanobacteria two different shapes of bubbles can be generated, resulting in bubbly flow or slug flow. Growth of Anabaena variabilis under slug flow (1.9g/l/day) was 1.73 times higher than that under bubbly flow (1.1g/l/day) when the specific irradiation rate was maintained above 10μmol/s/g dry cell. Although a stepwise increase in superficial gas velocity enhanced the average cell growth rate under bubbly flow by 1.57 times, the average cell growth rate during the deceleration phase under bubbly flow (1.98g/l/day) was 0.61 times smaller than that under slug flow (3.22g/l/day). These results demonstrate that the bubble shape in the slug flow was advantageous in regards to the radial circulation of cells.